Human tumor necrosis receptor TR9

ABSTRACT

The present invention relates to a novel member of the tumor necrosis factor family of receptors. In particular, isolated nucleic acid molecules are provided encoding the human TR9 receptor. TR9 polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of TR9 receptor activity.

This application is a divisional of U.S. application Ser. No.09/095,094, filed Jun. 10, 1998, which claims the benefit of the filingdate of Provisional Application No. 60/052,991, filed Jun. 11, 1997,each of which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a novel member of the tumor necrosisfactor family of receptors. More specifically, isolated nucleic acidmolecules are provided encoding a novel human tumor necrosis factorreceptor, TR9 (also known as Death Domain Containing Receptor 6, orsimply DR6). TR9 polypeptides are also provided, as are vectors, hostcells and recombinant methods for producing the same. The inventionfurther relates to screening methods for identifying agonists andantagonists of TR9 activity.

BACKGROUND OF THE INVENTION

Many biological actions, for instance, response to certain stimuli andnatural biological processes, are controlled by factors, such ascytokines. Many cytokines act through receptors by engaging the receptorand producing an intra-cellular response.

For example, tumor necrosis factors (TNF) alpha and beta are cytokines,which act through TNF receptors to regulate numerous biologicalprocesses, including protection against infection and induction of shockand inflammatory disease. The TNF molecules belong to the “TNF-ligand”superfamily, and act together with their receptors or counter-ligands,the “TNF-receptor” superfamily. So far, nine members of the TNF ligandsuperfamily have been identified and ten members of the TNF-receptorsuperfamily have been characterized.

Among the ligands there are included TNF-α, lymphotoxin-α (LT-α, alsoknown as TNF-β), LT-β (found in complex heterotrimer LT-α2-β), FasL,CD40L, CD27L, CD30L, 4-1BBL, OX40L and nerve growth factor (NGF). Thesuperfamily of TNF receptors includes the p55TNF receptor, p75TNFreceptor, TNF receptor-related protein, FAS antigen or APO-1, CD40,CD27, CD30, 4-1BB, OX40, low affinity p75 and NGF-receptor (Meager, A.,Biologicals 22:291-295 (1994)).

Many members of the TNF-ligand superfamily are expressed by activatedT-cells, implying that they are necessary for T-cell interactions withother cell types which underlie cell ontogeny and functions. (Meager,A., supra).

Considerable insight into the essential functions of several members ofthe TNF receptor family has been gained from the identification andcreation of mutants that abolish the expression of these proteins. Forexample, naturally occurring mutations in the FAS antigen and its ligandcause lymphoproliferative disease (Watanabe-Fukunaga. et al., Nature356:314 (1992)), perhaps reflecting a failure of programmed cell death.Mutations of the CD40 ligand cause an X-linked immunodeficiency statecharacterized by high levels of immunoglobulin M and low levels ofimmunoglobulin G in plasma, indicating faulty T-cell-dependent B-cellactivation (Allen et al., Science 259:990 (1993)). Targeted mutations ofthe low affinity nerve growth factor receptor cause a disordercharacterized by faulty sensory innovation of peripheral structures (Leeet al., Cell 69:737 (1992)).

TNF and LT-α are capable of binding to two TNF receptors (the 55- and75-kd TNF receptors). A large number of biological effects elicited byTNF and LT-∝, acting through their receptors, include hemorrhagicnecrosis of transplanted tumors, cytotoxicity, a role in endotoxicshock, inflammation, immunoregulation, proliferation and anti-viralresponses, as well as protection against the deleterious effects ofionizing radiation. TNF and LT-α are involved in the pathogenesis of awide range of diseases, including endotoxic shock, cerebral malaria,tumors, autoimmune disease, AIDS and graft-host rejection (Beutler etal., Science 264:667-668 (1994)). Mutations in the p55 receptor causeincreased susceptibility to microbial infection.

Moreover, an about 80 amino acid domain near the C-terminus of TNFR1(p55) and Fas was reported as the “death domain,” which is responsiblefor transducing signals for programmed cell death (Tartaglia et al.,Cell 74:845 (1993)).

Apoptosis, or programmed cell death, is a physiologic process essentialto the normal development and homeostasis of multicellular organisms(Steller, Science 267:1445-1449 (1995)). Derangements of apoptosiscontribute to the pathogenesis of several human diseases includingcancer, neurodegenerative disorders, and acquired immune deficiencysyndrome (Thompson C. B., Science 267:1456-1462 (1995)). Recently, muchattention has focused on the signal transduction and biological functionof two cell surface death receptors, Fas/APO-1 and TNFR-1 (Cleveland etal., Cell 81:479-482 (1995); Fraser et al., Cell 85:781-784 (1996); S.Nagata et al., Science 267:1449-56 (1995)). Both are members of the TNFreceptor family, which also include TNFR-2, low affinity NGFR, CD40, andCD30, among others (Smith et al., Science 248:1019-23 (1990); Tewari etal., in Modular Texts in Molecular and Cell Biology; M. Purton, Heldin,Carl, Ed. (Chapman and Hall, London, 1995). While family members aredefined by the presence of cysteine-rich repeats in their extracellulardomains, Fas/APO-1 and TNFR-1 also share a region of intracellularhomology, appropriately designated the “death domain,” which isdistantly related to the Drosophila suicide gene, reaper (Golstein etal., Cell 81:185-6 (1995); White et al., Science 264:677-83 (1994)).This shared death domain suggests that both receptors interact with arelated set of signal transducing molecules that, until recently,remained unidentified. Activation of Fas/APO-1 recruits the deathdomain-containing adapter molecule FADD/MORT1 (Chinnaiyan et al., Cell81:505-512 (1995); Boldin et al., J. Biol. Chem. 270:7795-8 (1995);Kischkel et al., EMBO 14:5579-5588 (1995)), which in turn binds andpresumably activates FLICE/MACH1, a member of the ICE/CED-3 family ofpro-apoptotic proteases (Muzio et al., Cell 85:817-827 (1996); Boldin etal., Cell 85:803-815 (1996)). While the central role of Fas/APO-1 is totrigger cell death, TNFR-1 can signal an array of diverse biologicalactivities-many of which stem from its ability to activate NF-kB(Tartaglia et al., Immunol Today 13:151-153 (1992)). Accordingly, TNFR-1recruits the multivalent adapter molecule TRADD, which like FADD, alsocontains a death domain (Hsu et al., Cell 81:495-504 (1995); Hsu et al.,Cell 84:299-308 (1996)). Through its associations with a number ofsignaling molecules including FADD, TRAF2, and RIP, TRADD can signalboth apoptosis and NF-kB activation (Hsu et al., Cell 84:299-308 (1996);Hsu et al., Immunity 4:387-396 (1996)).

The effects of TNF family ligands and receptors are varied and influencenumerous functions, both normal and abnormal, in the biologicalprocesses of the mammalian system. There is a clear need, therefore, foridentification and characterization of additional novel TNF receptorsand ligands that influence biological activity, both normally and indisease states.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the TR9 receptor having the aminoacid sequence shown in FIGS. 1A-D (SEQ ID NO:2) or the amino acidsequence encoded by the cDNA clone deposited as ATCC Deposit Number209037 on May 15, 1997.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofTR9 receptor polypeptides or peptides by recombinant techniques.

The invention further provides an isolated TR9 polypeptide having anamino acid sequence encoded by a polynucleotide described herein.

The present invention also provides a screening method for identifyingcompounds capable of enhancing or inhibiting a cellular response inducedby the TR9 receptor. The method involves contacting cells which expressthe TR9 receptor with the candidate compound, assaying a cellularresponse, and comparing the cellular response to a standard cellularresponse, the standard being assayed when contact is made in absence ofthe candidate compound; whereby, an increased cellular response over thestandard indicates that the compound is an agonist and a decreasedcellular response over the standard indicates that the compound is anantagonist.

The invention further provides diagnostic assays such as quantitativeand diagnostic assays for detecting levels of TR9 receptor protein.Thus, for instance, a diagnostic assay in accordance with the inventionfor detecting over-expression of TR9, or soluble form thereof, comparedto normal control tissue samples, may be used to detect the presence oftumors.

Tumor Necrosis Factor (TNF) family ligands are known to be among themost pleiotropic cytokines, inducing a large number of cellularresponses, including cytotoxicity, anti-viral activity, immunoregulatoryactivities, and the transcriptional regulation of several genes.Cellular response to TNF-family ligands include not only normalphysiological responses, but also diseases associated with increasedapoptosis or the inhibition of apoptosis. Apoptosis-programmed celldeath-is a physiological mechanism involved in the deletion ofperipheral T lymphocytes of the immune system, and its dysregulation canlead to a number of different pathogenic processes. Diseases associatedwith increased cell survival, or the inhibition of apoptosis, includecancers, autoimmune disorders, viral infections, inflammation, graft vs.host disease, acute graft rejection, and chronic graft rejection.Diseases associated with increased apoptosis include AIDS,neurodegenerative disorders, myelodysplastic syndromes, ischemic injury,toxin-induced liver disease, septic shock, cachexia, and anorexia.

Thus, the invention further provides a method for enhancing apoptosisinduced by a TNF-family ligand, which involves administering to a cellwhich expresses the TR9 polypeptide an effective amount of an agonistcapable of increasing TR9 mediated signaling. Preferably, TR9 mediatedsignaling is increased to treat a disease wherein decreased apoptosis isexhibited.

In a further aspect, the present invention is directed to a method forinhibiting apoptosis induced by a TNF-family ligand, which involvesadministering to a cell which expresses the TR9 polypeptide an effectiveamount of an antagonist capable of decreasing TR9 mediated signaling.Preferably, TR9 mediated signaling is decreased to treat a diseasewherein increased apoptosis is exhibited.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-D shows the nucleotide sequence (SEQ ID NO:1) and deduced aminoacid sequence (SEQ ID NO:2) of the TR9 receptor. Analysis using thecomputer program PSORT reveals that the protein has a predicted leadersequence of about 40 amino acid residues (underlined) and a deducedmolecular weight of about 72 kDa. It is further predicted that aminoacid residues from about 41 to about 350 constitute the extracellulardomain (amino acid residues from about 1 to about 310 in SEQ ID NO:2);from about 351 to about 367 the transmembrane domain (amino acidresidues from about 311 to about 327 in SEQ ID NO:2); from about 368 toabout 655 the intracellular domain (amino acid residues from about 328to about 615 in SEQ ID NO:2); and from about 429 to about 495 the deathdomain (amino acid residues from about 389 to about 455 in SEQ ID NO:2).

FIG. 2 shows the regions of similarity between the amino acid sequencesof the TR9 receptor protein and Fas (SEQ ID NO:3), NGFR p75 (SEQ IDNO:4), and TNFR 1 (SEQ ID NO:5).

FIG. 3 shows an analysis of the TR9 amino acid sequence. Alpha, beta,turn and coil regions; hydrophilicity and hydrophobicity; amphipathicregions; flexible regions; antigenic index and surface probability areshown. In the “Antigenic Index—Jameson-Wolf” graph, amino acid residuesabout 44 to about 121, about 156 to about 311, about 323 to about 348,about 376 to about 412, about 433 to about 474, about 485 to about 599,and about 611 to about 628 in FIGS. 1A-D correspond to the shown highlyantigenic regions of the TR9 protein. These highly antigenic fragmentsin FIGS. 1A-D correspond to the following fragments, respectively, inSEQ ID NO:2: amino acid residues about 4 to about 81, about 116 to about271, about 283 to about 308, about 336 to about 372, about 393 to about434, about 445 to about 559, and about 571 to about 588.

FIGS. 4A-C. Highlights of the predicted amino acid sequence of TR9. FIG.4A: The open reading frame for TR9 defines a type I transmembraneprotein of 655 amino acids (SEQ ID NO:2). Application of a computerprogram other than PSORT has predicted the mature protein to start atamino acid 42 (Gln, indicated by a black triangle). The putative signalpeptide and transmembrane domain are single and double underlined,respectively. Six potential N-glycosylation sites are indicated by blackdots. The cytoplasmic death domain is boxed. An intracellular regioncontaining a potential leucine-zipper motif overlapping with a prolinerich sequence is underlined with a thick line. FIG. 4B: Sequencealignment of extracellular cysteine-rich domains of TR9 (SEQ ID NO:19)and osteoprotegrin (SEQ ID NO:20). Alignment was done with Megalign(DNASTAR) software. Shading represents identical residues. FIG. 4C:Sequence comparison of death domains of TR9 (SEQ ID NO:21), CD95 (SEQ IDNO:22), TNFR1 (SEQ ID NO:23), DR3 (SEQ ID NO:24), DR4 (SEQ ID NO:25),and DR5 (SEQ ID NO:26). Alignment was performed and represented in thesame way as in FIG. 4B. OPG; osteoprotegerin.

FIG. 5. TR9 induces apoptosis in mammalian cells. Ectopic expression ofTR9 induces apoptosis in Hela cells, but not in MCF7 cells. Hela andMCF7 cells were cotransfected with a empty vector, TR9, TR9 delta, orDR4, together with a β-galactosidase-expressing reporter construct usinga lipofectamine method according to the manufacturer's instructions(BRL). Nineteen hours after transfection, cells were stained with5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside (X-Gal) and examined asdescribed in Chinnaiyan et al., Cell 81:505-512 (1995). The data(mean±SD) represent the percentage of round, apoptotic cells as afunction of total β-galactosidase-positive cells (n=4).

FIG. 6. TR9 mediates nuclear factor kB activation. Cotransfection of 293cells was performed with the indicated expression constructs and a NF-kBluciferase reporter construct. After transfection (at 36 hours), cellextracts were prepared and luciferase activities determined aspreviously described (Chinnaiyan et al., Science 274:990-992 (1996); andPan et al., Science 276:111-113 (1997)). Transfection efficiency wasmonitored by β-galactosidase activity. A portion of the transfectedcells was used to monitor expression of TR9 or TR9 delta. Cell lysateswere prepared and immunoprecipitated with FLAG M2 affinity gel and thepresence of TR9 or TR9 delta detected by blotting with anti-FLAG.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding a TR9 receptor polypeptide havingthe amino acid sequence shown in FIGS. 1A-C (SEQ ID NO:2), which wasdetermined by sequencing a cloned cDNA. As shown in FIG. 2, the TR9receptor protein of the present invention shares sequence homology withFas (SEQ ID NO:3), NGFR p75 (SEQ ID NO:4), and TNFR 1(SEQ ID NO:5). Thenucleotide sequence shown in SEQ ID NO:1 was obtained by sequencing acDNA clone, which was deposited on May 15, 1997 at the American TypeCulture Collection, 12301 Park Lawn Drive, Rockville, Md. 20852, andgiven accession number 209037. The deposited clone is inserted in thepBluescript SK(−) plasmid (Stratagene, LaJolla, Calif.) using the EcoRIand XhoI restriction endonuclease cleavage sites.

Nucleic Acid Molecules

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc.), and allamino acid sequences of polypeptides encoded by DNA molecules determinedherein were predicted by translation of a DNA sequence determined asabove. Therefore, as is known in the art for any DNA sequence determinedby this automated approach, any nucleotide sequence determined hereinmay contain some errors. Nucleotide sequences determined by automationare typically at least about 90% identical, more typically at leastabout 95% to at least about 99.9% identical to the actual nucleotidesequence of the sequenced DNA molecule. The actual sequence can be moreprecisely determined by other approaches including manual DNA sequencingmethods well known in the art. As is also known in the art, a singleinsertion or deletion in a determined nucleotide sequence compared tothe actual sequence will cause a frame shift in translation of thenucleotide sequence such that the predicted amino acid sequence encodedby a determined nucleotide sequence will be completely different fromthe amino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

Using the information provided herein, such as the nucleotide sequencein FIGS. 1A-D (SEQ ID NO:1), a nucleic acid molecule of the presentinvention encoding a TR9 polypeptide may be obtained using standardcloning and screening procedures, such as those for cloning cDNAs usingmRNA as starting material. Illustrative of the invention, the nucleicacid molecule described in FIGS. 1A-D (SEQ ID NO:1) was discovered in acDNA library derived from human microvascular endothelial cells. Thegene was also identified in cDNA libraries from the following tissues:human placenta, stromal cells, human amygdala, human umbilical veinendothelial cells, kidney cancer, human gall bladder, soares adultbrain, normal human liver, hepatocellular tumor, keratinocytes, bonemarrow, macrophage, human synovial sarcoma, human hippocampus, and humantonsils.

The determined nucleotide sequence of the TR9 cDNA of FIGS. 1A-D (SEQ IDNO:1) contains an open reading frame encoding a protein of about 615amino acid residues, with a predicted leader sequence of about 40 aminoacid residues, and a deduced molecular weight of about 72 kDa. The aminoacid sequence of the predicted mature TR9 receptor is shown in FIGS.1A-D (SEQ ID NO:2) from amino acid residue about 1 to residue about 615.The TR9 protein shown in FIGS. 1A-D (SEQ ID NO:2) is about 24% identicaland about 43% similar to NGFR (FIG. 2).

As predicted by the sequence homology exhibited between TR9 and otherdeath domain containing receptors (see FIG. 4C), TR9 induces ofmammalian cells apoptosis (see FIG. 6). It is expected that TR9-inducedapoptosis will be efficiently blocked by inhibitors of death proteasesincluding z-VAD-fmk, an irreversible broad spectrum caspase inhibitorand CrmA, a cowpox virus encoded serpin that preferentially inhibitsapical caspases such as FLICE/MACH-1 (caspase-8).

As indicated, the present invention also provides the mature form(s) ofthe TR9 receptor of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal orsecretory leader sequence which is cleaved from the mature protein onceexport of the growing protein chain across the rough endoplasmicreticulum has been initiated. Most mammalian cells and even insect cellscleave secreted proteins with the same specificity. However, in somecases, cleavage of a secreted protein is not entirely uniform, whichresults in two or more mature species on the protein. Further, it haslong been known that the cleavage specificity of a secreted protein isultimately determined by the primary structure of the complete protein,that is, it is inherent in the amino acid sequence of the polypeptide.Therefore, the present invention provides a nucleotide sequence encodingthe mature TR9 receptor polypeptides having the amino acid sequenceencoded by the cDNA clone contained in the host identified as ATCCDeposit No. 209037 and as shown in FIGS. 1A-D (SEQ ID NO:2). By themature TR9 protein having the amino acid sequence encoded by the cDNAclone contained in the host identified as ATCC Deposit 209037 is meantthe mature form(s) of the TR9 receptor produced by expression in amammalian cell (e.g., COS cells, as described below) of the completeopen reading frame encoded by the human DNA sequence of the clonecontained in the vector in the deposited host. As indicated below, themature TR9 receptor having the amino acid sequence encoded by the cDNAclone contained in ATCC Deposit No. 209037 may or may not differ fromthe predicted “mature” TR9 receptor protein shown in SEQ ID NO:2 (aminoacids from about 1 to about 615) depending on the accuracy of thepredicted cleavage site based on computer analysis.

Methods for predicting whether a protein has a secretory leader as wellas the cleavage point for that leader sequence are available. Forinstance, the methods of McGeoch (Virus Res. 3:271-286 (1985)) and vonHeinje (Nucleic Acids Res. 14:4683-4690 (1986)) can be used. Theaccuracy of predicting the cleavage points of known mammalian secretoryproteins for each of these methods is in the range of 75-80%. vonHeinje, supra. However, the two methods do not always produce the samepredicted cleavage point(s) for a given protein.

In the present case, the predicted amino acid sequence of the completeTR9 polypeptides of the present invention were analyzed by a computerprogram (“PSORT”) (K. Nakai and M. Kanehisa, Genomics 14:897-911(1992)), which is an expert system for predicting the cellular locationof a protein based on the amino acid sequence. As part of thiscomputational prediction of localization, the methods of McGeoch and vonHeinje are incorporated. The analysis by the PSORT program predicted thecleavage site between amino acid residues 40 and 41 in FIGS. 1A-D (aminoacid residues −1 and 1 in SEQ ID NO:2). Thereafter, the complete aminoacid sequences were further analyzed by visual inspection, applying asimple form of the (−1,−3) rule of von Heinje. von Heinje, supra. Thus,the leader sequence for the TR9 receptor protein is predicted to consistof amino acid residues from about 1 to 40 in FIGS. 1A-D (amino acidresidues −40 to about −1 in SEQ ID NO:2), while the mature TR9 proteinis predicted to consist of residues from about 41 to 655 in FIGS. 1A-D(about 1 to about 615 of SEQ ID NO:2). Analysis using a differentcomputer program predicts that the mature protein of TR9 starts at aminoacid 42 (Gln) as depicted in FIGS. 1A-D and 4A. The results of thisanalysis are presented in FIG. 4A and described in Example 6.

As one of ordinary skill would appreciate, due to the possibility ofsequencing errors, as well as the variability of cleavage sites forleaders in different known proteins, the predicted TR9 receptorpolypeptide encoded by the deposited cDNA comprises about 655 aminoacids, but may be anywhere in the range of 645-665 amino acids; and thepredicted leader sequence of this protein is about 40 amino acids, butmay be anywhere in the range of about 30 to about 50 amino acids.

As indicated, nucleic acid molecules of the present invention may be inthe form of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

Isolated nucleic acid molecules of the present invention include DNAmolecules comprising an open reading frame (ORF) shown in FIGS. 1A-D(SEQ ID NO:1); DNA molecules comprising the coding sequence for themature TR9 protein; and DNA molecules which comprise a sequencesubstantially different from those described above but which, due to thedegeneracy of the genetic code, still encode the TR9 protein shown inFIGS. 1A-D (SEQ ID NO:2). Of course, the genetic code is well known inthe art. Thus, it would be routine for one skilled in the art togenerate such degenerate variants.

In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of the nucleotidesequence in FIGS. 1A-D of (SEQ ID NO:1), which have been determined fromthe following related cDNA clones: HIBEJ86R (SEQ ID NO:6), HL1AA79R (SEQID NO:7), HHFGD57R (SEQ ID NO:8), HSABG38R (SEQ ID NO:9), and HHPDZ31R(SEQ ID NO:10).

Further, the invention includes a polynucleotide comprising any portionof at least about 30 nucleotides, preferably at least about 50nucleotides, of the nucleotide sequence disclosed in FIGS. 1A-D fromnucleotides 655 to 907 (nucleotides 615 to 867 of SEQ ID NO:1) and/orthe nucleotide sequence disclosed in FIGS. 1A-D from nucleotides to 540to 1020 (nucleotides 500 to 980 as depicted in SEQ ID NO:1).

In another aspect, the invention provides isolated nucleic acidmolecules encoding the TR9 receptor polypeptide having an amino acidsequence as encoded by the cDNA clone contained in the plasmid depositedas ATCC Deposit No. 209037 on May 15, 1997. In a further embodiment,nucleic acid molecules are provided encoding the mature TR9 receptorpolypeptide or the full-length TR9 receptor polypeptide lacking theN-terminal methionine. The invention also provides an isolated nucleicacid molecule having the nucleotide sequence shown in FIGS. 1A-D (SEQ IDNO:1) or the nucleotide sequence of the TR9 cDNA contained in theabove-described deposited clone, or a nucleic acid molecule having asequence complementary to one of the above sequences. Such isolatedmolecules, particularly DNA molecules, are useful as probes for genemapping, by in situ hybridization with chromosomes, and for detectingexpression of the TR9 receptor gene in human tissue, for instance, byNorthern blot analysis.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatednucleic acid molecule having the nucleotide sequence of the depositedcDNA, or the nucleotide sequence shown in FIGS. 1A-D (SEQ ID NO:1), orthe complementary strand thereto, is intended fragments at least about15 nt, and more preferably at least about 20 nt, still more preferablyat least about 30 nt, and even more preferably, at least about 40, 50,100, 150, 200, 250, 300, 400, or 500 nt in length. These fragments havenumerous uses which include, but are not limited to, diagnostic probesand primers as discussed herein. Of course, larger fragments 50-1500 ntin length are also useful according to the present invention as arefragments corresponding to most, if not all, of the nucleotide sequenceof the deposited cDNA or as shown in FIGS. 1A-D (SEQ ID NO:1). By afragment at least 20 nt in length, for example, is intended fragmentswhich include 20 or more contiguous bases from the nucleotide sequenceof the deposited cDNA or the nucleotide sequence as shown in FIGS. 1A-D(SEQ ID NO:1).

Representative examples of TR9 polynucleotide fragments of the inventioninclude, for example, fragments that comprise, or alternatively, consistof, a sequence from about nucleotide 1-50, 51-100, 101-150, 151-200,201-250, 251-300, 301-350, 351-400, 401-450, 445-879, 451-500, 501-550,551-600, 615-651, 651-700, 701-750, 751-800, 800-850, 850-867, 851-900,901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200,1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500,1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800,1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-3000, or3001 to the end of SEQ ID NO:1, or the complementary DNA strand thereto,or the cDNA contained in the deposited clone. In this context “about”includes the particularly recited ranges, larger or smaller by several(5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.In specific embodiments, the polynucleotide fragments of the inventionencode a polypeptide which demonstrates a functional activity. By apolypeptide demonstrating “functional activity” is meant, a polypeptidecapable of displaying one or more known functional activities associatedwith a complete or mature TR9 polypeptide. Such functional activitiesinclude, but are not limited to, biological activity, antigenicity[ability to bind (or compete with a TR9 polypeptide for binding) to ananti-TR9 antibody], immunogenicity (ability to generate antibody whichbinds to a TR9 polypeptide), and ability to bind to a receptor or ligandfor a TR9 polypeptide.

Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding: a polypeptide comprising the TR9receptor extracellular domain (predicted to constitute amino acidresidues from about 1 to about 310 in SEQ ID NO:2); a polypeptidecomprising the four TNFR-like cysteine rich motifs of TR9 (amino acidresidues 67 to 211 in FIGS. 1A-D; amino acid residues 27 to 171 in SEQID NO:2), a polypeptide comprising the TR9 receptor transmembrane domain(predicted to constitute amino acid residues from about 311 to about 327in SEQ ID NO:2); a polypeptide comprising the TR9 receptor intracellulardomain (predicted to constitute amino acid residues from about 328 toabout 615 in SEQ ID NO:2); a polypeptide comprising the TR9 receptorextracellular and intracellular domains with all or part of thetransmembrane domain deleted; a polypeptide comprising the TR9 receptordeath domain (predicted to constitute amino acid residues from about 389to about 455 in SEQ ID NO:2); and nucleic acid molecules encodingepitope bearing portions of the TR9 receptor protein. As above, with theleader sequence, the amino acid residues constituting the TR9 receptorextracellular, transmembrane and intracellular domains have beenpredicted by computer analysis. Thus, as one of ordinary skill wouldappreciate, the amino acid residues constituting these domains may varyslightly (e.g., by about 1 to about 15 amino acid residues) depending onthe criteria used to define each domain.

Preferred nucleic acid fragments of the present invention also includenucleic acid molecules encoding one or more epitope-bearing portions ofthe TR9 receptor protein. In particular, such nucleic acid fragments ofthe present invention include nucleic acid molecules encoding: apolypeptide comprising amino acid residues from about 4 to about 81 inSEQ ID NO:2; a polypeptide comprising amino acid residues from about 116to about 271 in SEQ ID NO:2; a polypeptide comprising amino acidresidues from about 283 to about 308 in SEQ ID NO:2; a polypeptidecomprising amino acid residues from about 336 to about 372 in SEQ IDNO:2; a polypeptide comprising amino acid residues from about 393 toabout 434 in SEQ ID NO:2; a polypeptide comprising amino acid residuesfrom about 445 to about 559 in SEQ ID NO:2; and a polypeptide comprisingamino acid residues from about 571 to about 588 in SEQ ID NO:2. Theinventors have determined that the above polypeptide fragments areantigenic regions of the TR9 receptor. Methods for determining othersuch epitope-bearing portions of the TR9 protein are described in detailbelow.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes, preferably understringent hybridization conditions, to a portion of the polynucleotidesequence of a polynucleotide of the invention such as, for instance, thecDNA clone contained in ATCC Deposit 209037. By “stringent hybridizationconditions” is intended overnight incubation at 42° C. in a solutioncomprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 g/nml denatured, sheared salmon sperm DNA, followed bywashing the filters in 0.1×SSC at about 65° C.

By a polynucleotide which hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30-70, or 80-150 nt, or the entire length of the referencepolynucleotide. These are useful as diagnostic probes and primers asdiscussed above and in more detail below.

In specific embodiments, polynucleotides of the invention hybridize to acomplementary strand of a polynucleotide encoding amino acid residues40-152, 40-48, 40-51, 51-66, 66-73, 73-83, 83-104, 104-110, 110-128,128-146, and/or 146-152 as depicted in SEQ ID NO:2.

By a portion of a polynucleotide of “at least 20 nt in length,” forexample, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNA or the nucleotide sequence as shown in FIGS. 1A-D (SEQ ID NO:1).

Of course, a polynucleotide which hybridizes only to a poly A sequence(such as the 3′ terminal poly tract of the TR9 receptor cDNA shown inSEQ ID NO:1), or to a complementary stretch of T (or U) residues, wouldnot be included in a polynucleotide of the invention used to hybridizeto a portion of a nucleic acid of the invention, since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone generated using oligo dT as a primer).

As indicated, nucleic acid molecules of the present invention whichencode a TR9 receptor polypeptide may include, but are not limited to,those encoding the amino acid sequence of the mature polypeptide, byitself; the coding sequence for the mature polypeptide and additionalsequences, such as those encoding the about amino acid leader orsecretory sequence, such as a pre-, or pro- or prepro-protein sequence;the coding sequence of the mature polypeptide, with or without theaforementioned additional coding sequences, together with additional,non-coding sequences, including for example, but not limited to intronsand non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription, mRNAprocessing, including splicing and polyadenylation signals, forexample—ribosome binding and stability of mRNA; an additional codingsequence which codes for additional amino acids, such as those whichprovide additional functionalities. Thus, the sequence encoding thepolypeptide may be fused to a marker sequence, such as a sequenceencoding a peptide which facilitates purification of the fusedpolypeptide. In certain preferred embodiments of this aspect of theinvention, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (Qiagen, Inc.), among others,many of which are commercially available. As described in Gentz et al.,Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. The “HA” tag is another peptide useful for purification whichcorresponds to an epitope derived from the influenza hemagglutininprotein, which has been described by Wilson et al., Cell 37:767-778(1984). As discussed below, other such fusion proteins include the TR9receptor fused to Fc at the N— or C-terminus.

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of the TR9 receptor. Variants may occur naturally, such as anatural allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions, which may involve one or more nucleotides. Thevariants may be altered in coding regions, non-coding regions, or both.Alterations in the coding regions may produce conservative ornon-conservative amino acid substitutions, deletions or additions.Especially preferred among these are silent substitutions, additions anddeletions, which do not alter the properties and activities of the TR9receptor or portions thereof. Also especially preferred in this regardare conservative substitutions.

Further embodiments of the invention include isolated nucleic acidmolecules comprising a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98% or99% identical to (a) a nucleotide sequence encoding the polypeptidehaving the amino acid sequence shown in FIGS. 1A-D (SEQ ID NO:2); (b) anucleotide sequence encoding the polypeptide having the amino acidsequence shown in FIGS. 1A-D (SEQ ID NO:2), but lacking the N-terminalmethionine; (c) a nucleotide sequence encoding the predicted mature TR9polypeptide (full-length polypeptide with any attending leader sequenceremoved) comprising the amino acid sequence at positions from about 1 toabout 615 in SEQ ID NO:2; (d) a nucleotide sequence encoding the TR9polypeptide having the amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 209037; (e) a nucleotide sequence encodingthe mature TR9 polypeptide having the amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No. 209037; (f) a nucleotidesequence encoding the TR9 receptor extracellular domain; (g) anucleotide sequence encoding the four TNFR-like cysteine rich motifs ofTR9 (amino acid residues 67 to 211 in FIGS. 1A-D; amino acid residues 27to 171 in SEQ ID NO:2); (h) a nucleotide sequence encoding the TR9receptor transmembrane domain; (i) a nucleotide sequence encoding theTR9 receptor intracellular domain; (j) a nucleotide sequence encodingthe TR9 receptor extracellular and intracellular domains with all orpart of the transmembrane domain deleted; (k) a nucleotide sequenceencoding the TR9 receptor death domain; (l) a nucleotide sequenceencoding the TR9 leucine zipper; and (m) a nucleotide sequencecomplementary to any of the nucleotide sequences in (a), (b), (c), (d),(e), (f), (g), (h), (i), (j), (k), or (l).

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a TR9receptor polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the TR9receptor. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto 5% of the nucleotides in the reference sequence may be deleted orsubstituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence may be inserted intothe reference sequence. These mutations of the reference sequence mayoccur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among nucleotides in the reference sequence or inone or more contiguous groups within the reference sequence. Thereference (query) sequence may be the entire TR9 nucleotide sequenceshown in FIGS. 1A-D (SEQ ID NO:1) or any fragment as described herein.

As a practical matter, whether any particular nucleic acid molecule isat least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, thenucleotide sequence shown in FIGS. 1A-D (SEQ ID NO:1) or to thenucleotides sequence of the deposited cDNA clone can be determinedconventionally using known computer programs such as the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2:482-489 (1981), to find thebest segment of homology between two sequences. When using Bestfit orany other sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the fill lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDBalignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter. According to this embodiment, if thesubject sequence is shorter than the query sequence because of 5′ or 3′deletions, not because of internal deletions, a manual correction ismade to the results to take into consideration the fact that the FASTDBprogram does not account for 5′ and 3′ truncations of the subjectsequence when calculating percent identity. For subject sequencestruncated at the 5′ or 3′ ends, relative to the query sequence, thepercent identity is corrected by calculating the number of bases of thequery sequence that are 5′ and 3′ of the subject sequence, which are notmatched/aligned, as a percent of the total bases of the query sequence.A determination of whether a nucleotide is matched/aligned is determinedby results of the FASTDB sequence alignment. This percentage is thensubtracted from the percent identity, calculated by the above FASTDBprogram using the specified parameters, to arrive at a final percentidentity score. This corrected score is what is used for the purposes ofthis embodiment. Only bases outside the 5′ and 3′ bases of the subjectsequence, as displayed by the FASTDB alignment, which are notmatched/aligned with the query sequence, are calculated for the purposesof manually adjusting the percent identity score. For example, a 90 basesubject sequence is aligned to a 100 base query sequence to determinepercent identity. The deletions occur at the 5′ end of the subjectsequence and therefore, the FASTDB alignment does not show amatched/alignment of the first 10 bases at 5′ end. The 10 unpaired basesrepresent 10% of the sequence (number of bases at the 5′ and 3′ ends notmatched/total number of bases in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram If the remaining 90 bases were perfectly matched the finalpercent identity would be 90%. In another example, a 90 base subjectsequence is compared with a 100 base query sequence. This time thedeletions are internal deletions so that there are no bases on the 5′ or3′ of the subject sequence which are not matched/aligned with the query.In this case the percent identity calculated by FASTDB is not manuallycorrected. Once again, only bases 5′ and 3′ of the subject sequencewhich are not matched/aligned with the query sequence are manuallycorrected for. No other manual corrections are made for the purposes ofthis embodiment.

The present application is directed to nucleic acid molecules at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in FIGS. 1A-D (SEQ ID NO:1) or to the nucleic acid sequence of thedeposited cDNA, irrespective of whether they encode a polypeptide havingTR9 receptor activity. This is because even where a particular nucleicacid molecule does not encode a polypeptide having TR9 receptoractivity, one of skill in the art would still know how to use thenucleic acid molecule, for instance, as a hybridization probe or apolymerase chain reaction (PCR) primer. Uses of the nucleic acidmolecules of the present invention that do not encode a polypeptidehaving TR9 receptor activity include, inter alia, (1) isolating the TR9receptor gene or allelic variants thereof in a cDNA library; (2) in situhybridization (e.g., “FISH”) to metaphase chromosomal spreads to provideprecise chromosomal location of the TR9 receptor gene, as described inVerma et al., Human Chromosomes: A Manual of Basic Techniques, PergamonPress, N.Y. (1988); and (3) Northern Blot analysis for detecting TR9receptor mRNA expression in specific tissues.

Preferred, however, are nucleic acid molecules having sequences at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in FIGS. 1A-D (SEQ ID NO:1), the nucleic acid sequence of thedeposited cDNA, or fragments thereof, which do, in fact, encode apolypeptide having TR9 receptor activity. By “a polypeptide having TR9receptor activity” is intended polypeptides exhibiting activity similar,but not necessarily identical, to an activity of the TR9 receptor of theinvention (either the full-length protein or, preferably, the matureprotein), as measured in a particular immunoassay and/or biologicalassay. For example, TR9 receptor activity can be measured using the celldeath assays performed essentially as previously described (Chinnaiyanet al., Cell 81:505-512 (1995); Boldin et al., J. Biol. Chem.270:7795-8(1995); Kischkel et al., EMBO 14:5579-5588 (1995); Chinnaiyanet al., J. Biol. Chem. 271:4961-4965 (1996)) and as set forth in Example5 below. In MCF7 cells, plasmids encoding full-length TR9 or a candidatedeath domain containing receptor are co-transfected with the pLanternreporter construct encoding green fluorescent protein. Nuclei of cellstransfected with TR9 will exhibit apoptotic morphology as assessed byDAPI staining. It is expected that like TNFR-1 and Fas/APO-1 (Muzio etal., Cell 85:817-827 (1996); Boldin et al., Cell 85:803-815 (1996);Tewari et al., J. Biol. Chem. 270:3255-60 (1995)), TR9-induced apoptosiswill be blocked by the inhibitors of ICE-like proteases, CrmA andz-VAD-fmk. In addition, it is expected that apoptosis induced by TR9will be blocked by dominant negative versions of FADD (FADD-DN) or FLICE(FLICE-DN/MACHa1C360S).

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%,98%, or 99% identical to the nucleic acid sequence of the deposited cDNAor the nucleic acid sequence shown in FIGS. 1A-D (SEQ ID NO:1), orfragments thereof, will encode a polypeptide “having TR9 receptoractivity.” In fact, since degenerate variants of these nucleotidesequences all encode the same polypeptide, in many instances, this willbe clear to the skilled artisan even without performing the abovedescribed assay. It will be further recognized in the art that, for suchnucleic acid molecules that are not degenerate variants, a reasonablenumber will also encode a polypeptide having TR9 receptor activity. Thisis because the skilled artisan is fully aware of amino acidsubstitutions that are either less likely or not likely to significantlyeffect protein function (e.g., replacing one aliphatic amino acid with asecond aliphatic amino acid).

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in J. U. Bowie et al., “Deciphering theMessage in Protein Sequences: Tolerance to Amino Acid Substitutions,”Science 247:1306-1310 (1990), wherein the authors indicate that proteinsare surprisingly tolerant of amino acid substitutions.

Polynucleotide Assays

This invention is also related to the use of TR9 polynucleotides todetect complementary polynucleotides such as, for example, as adiagnostic reagent. Detection of a mutated form of TR9 associated with adysfunction will provide a diagnostic tool that can add or define adiagnosis of a disease or susceptibility to a disease which results fromunder-expression over-expression or altered expression of TR9 or asoluble form thereof, such as, for example, tumors or autoimmunedisease.

Individuals carrying mutations in the TR9 gene may be detected at theDNA level by a variety of techniques. Nucleic acids for diagnosis may beobtained from a patient's cells, such as from blood, urine, saliva,tissue biopsy and autopsy material. The genomic DNA may be used directlyfor detection or may be amplified enzymatically by using PCR prior toanalysis. (Saiki et al., Nature 324:163-166 (1986)). RNA or cDNA mayalso be used in the same ways. As an example, PCR primers complementaryto the nucleic acid encoding TR9 can be used to identify and analyze TR9expression and mutations. For example, deletions and insertions can bedetected by a change in size of the amplified product in comparison tothe normal genotype. Point mutations can be identified by hybridizingamplified DNA to radiolabeled TR9 RNA or alternatively, radiolabeled TR9antisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

Sequence differences between a reference gene and genes having mutationsalso may be revealed by direct DNA sequencing. In addition, cloned DNAsegments may be employed as probes to detect specific DNA segments. Thesensitivity of such methods can be greatly enhanced by appropriate useof PCR or another amplification method. For example, a sequencing primeris used with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels, with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science 230:1242 (1985)).

Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci. USA85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,restriction fragment length polymorphisms (“RFLP”) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations also can be detected by in situ analysis.

Vectors and Host Cells

The present invention also relates to vectors which include the isolatedDNA molecules of the present invention, host cells which are geneticallyengineered with the recombinant vectors, or which are otherwiseengineered to produce the polypeptides of the invention, and theproduction of TR9 receptor polypeptides, or fragments thereof, byrecombinant techniques.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

In one embodiment, the DNA of the invention is operatively associatedwith an appropriate heterologous regulatory element (e.g., promoter orenhancer), such as, the phage lambda PL promoter, the E. coli lac, trp,and tac promoters, the SV40 early and late promoters and promoters ofretroviral LTRs, to name a few. Other suitable promoters will be knownto the skilled artisan.

In embodiments in which vectors contain expression constructs, theseconstructs will further contain sites for transcription initiation,termination and, in the transcribed region, a ribosome binding site fortranslation. The coding portion of the mature transcripts expressed bythe constructs will preferably include a translation initiating at thebeginning and a termination codon (UAA, UGA or UAG) appropriatelypositioned at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase orneomycin resistance for eukaryotic cell culture and tetracycline orampicillin resistance genes for culturing in E. coli and other bacteria.Representative examples of appropriate hosts include, but are notlimited to, bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells; and plant cells. Appropriateculture mediums and conditions for the above-described host cells areknown in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia. Other suitable vectors will be readilyapparent to the skilled artisan.

Selection of appropriate vectors and promoters for expression in a hostcell is a well known procedure and the requisite techniques forexpression vector construction, introduction of the vector into the hostand expression in the host are routine skills in the art.

The present invention also relates to host cells containing the vectorconstructs discussed herein, and additionally encompasses host cellscontaining nucleotide sequences of the invention that are operablyassociated with one or more heterologous control regions (e.g., promoterand/or enhancer) using techniques known of in the art. The host cell canbe a higher eukaryotic cell, such as a mammalian cell (e.g., a humanderived cell), or a lower eukaryotic cell, such as a yeast cell, or thehost cell can be a prokaryotic cell, such as a bacterial cell. The hoststrain may be chosen which modulates the expression of the inserted genesequences, or modifies and processes the gene product in the specificfashion desired. Expression from certain promoters can be elevated inthe presence of certain inducers; thus expression of the geneticallyengineered polypeptide may be controlled. Furthermore, different hostcells have characteristics and specific mechanisms for the translationaland post-translational processing and modification (e.g.,phosphorylation, cleavage) of proteins. Appropriate cell lines can bechosen to ensure the desired modifications and processing of the foreignprotein expressed.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

The polypeptide may be expressed in a modified form, such as a fusionprotein (comprising the polypeptide joined via a peptide bond to aheterologous protein sequence (of a different protein)), and may includenot only secretion signals, but also additional heterologous functionalregions. Such a fusion protein can be made by ligating polynucleotidesof the invention and the desired nucleic acid sequence encoding thedesired amino acid sequence to each other, by methods known in the art,in the proper reading frame, and expressing the fusion protein productby methods known in the art. Alternatively, such a fusion protein can bemade by protein synthetic techniques, e.g., by use of a peptidesynthesizer. Thus, for instance, a region of additional amino acids,particularly charged amino acids, may be added to the N-terminus of thepolypeptide to improve stability and persistence in the host cell,during purification, or during subsequent handling and storage.Additionally, peptide moieties may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art.

A preferred fusion protein comprises a heterologous region fromimmunoglobulin that is useful to solubilize proteins. For example,EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteinscomprising various portions of constant region of immunoglobin moleculestogether with another human protein or part thereof. In many cases, theFc part in a fusion protein is thoroughly advantageous for use intherapy and diagnosis and thus results, for example, in improvedpharmacokinetic properties (EPA 0 232 262). On the other hand, for someuses it would be desirable to be able to delete the Fc part after thefusion protein has been expressed, detected and purified in theadvantageous manner described. This is the case when the Fc portionproves to be a hindrance to use in therapy and diagnosis, for examplewhen the fusion protein is to be used as an antigen for immunizations.In drug discovery, for example, human proteins, such as thehIL5-receptor, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,Bennett et al., J. of Molec. Recognition 8:52-58 (1995) and Johanson etal., J. Biol. Chem. 270:9459-9471 (1995).

The TR9 receptor can be recovered and purified from recombinant cellcultures by standard methods which include, but are not limited to,ammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography and lectin chromatography. Mostpreferably, high performance liquid chromatography (“HPLC”) is employedfor purification.

Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue oralternatively, may be missing the N-terminal methionine, in some casesas a result of host-mediated processes.

TR9 receptor polynucleotides and polypeptides may be used in accordancewith the present invention for a variety of applications, particularlythose that make use of the chemical and biological properties of TR9.Among these are applications in treatment of tumors, resistance toparasites, bacteria and viruses, to induce proliferation of T-cells,endothelial cells and certain hematopoietic cells, to treat restenosis,graft vs. host disease, to regulate anti-viral responses and to preventcertain autoimmune diseases after stimulation of TR9 by an agonist.Additional applications relate to diagnosis and to treatment ofdisorders of cells, tissues and organisms. These aspects of theinvention are discussed further below.

TR9 Receptor Polypeptides and Fragments

The invention further provides an isolated TR9 receptor polypeptidehaving the amino acid sequence encoded by the deposited cDNA, or theamino acid sequence in FIGS. 1A-D (SEQ ID NO:2), or a peptide orpolypeptide comprising a portion of the above polypeptides.

The polypeptides of this invention may be membrane bound or may be in asoluble circulating form. Soluble peptides are defined by amino acidsequence wherein the sequence comprises the polypeptide sequence lackingthe transmembrane domain.

The polypeptides of the present invention may exist as a membrane boundreceptor having a transmembrane region and an intra- and extracellularregion or they may exist in soluble form wherein the transmembranedomain is lacking. One example of such a form of the TR9 receptor is theTR9 receptor shown in FIGS. 1A-D (SEQ ID NO:2) which contains, inaddition to a leader sequence, transmembrane, intracellular andextracellular domains. Thus, this form of the TR9 receptor appears to belocalized in the cytoplasmic membrane of cells which express thisprotein.

It will be recognized in the art that some amino acid sequences of theTR9 receptor can be varied without significant effect on the structureor function of the protein. If such differences in sequence arecontemplated, it should be remembered that there will be critical areason the protein which determine activity. Thus, the invention furtherincludes variations of the TR9 receptor which show substantial TR9receptor activity or which include regions of TR9 receptor protein suchas the protein portions discussed below. Such mutants include deletions,insertions, inversions, repeats, and type substitutions. As indicatedabove, guidance concerning which amino acid changes are likely to bephenotypically silent can be found in Bowie et al., “Deciphering theMessage in Protein Sequences: Tolerance to Amino Acid Substitutions,”Science 247:1306-1310 (1990).

Thus, the fragment, derivative or analog of the polypeptide of FIGS.1A-D (SEQ ID NO:2), or that encoded by the deposited cDNA, may be: (i)one in which one or more of the amino acid residues are substituted witha conserved or non-conserved amino acid residue (preferably a conservedamino acid residue(s), and more preferably at least one but less thanten conserved amino acid residues), and such substituted amino acidresidue(s) may or may not be one encoded by the genetic code; or (ii)one in which one or more of the amino acid residues includes asubstituent group; or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol); or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asan IgG Fc fusion region peptide or leader or secretory sequence or asequence which is employed for purification of the mature polypeptide ora proprotein sequence. Such fragments, derivatives and analogs aredeemed to be within the scope of those skilled in the art from theteachings herein.

Of particular interest are substitutions of charged amino acids withanother charged amino acid and with neutral or negatively charged aminoacids. The latter results in proteins with reduced positive charge toimprove the characteristics of the TR9 receptor. The prevention ofaggregation is highly desirable. Aggregation of proteins not onlyresults in a loss of activity but can also be problematic when preparingpharmaceutical formulations, because they can be immunogenic. (Pinckardet al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes36:838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug CarrierSystems 10:307-377 (1993)).

The replacement of amino acids can also change the selectivity ofbinding to cell surface receptors. Ostade et al., Nature 361:266-268(1993), describes certain mutations resulting in selective binding ofTNF-α to only one of the two known types of TNF receptors. Thus, the TR9receptor of the present invention may include one or more amino acidsubstitutions, deletions, or additions, either from natural mutations orhuman manipulation.

As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table 1). TABLE 1Conservative Amino Acid Substitutions Aromatic Phenylalanine TryptophanTyrosine Hydrophobic Leucine Isoleucine Valine Polar GlutamineAsparagine Basic Arginine Lysine Histidine Acidic Aspartic Acid GlutamicAcid Small Alanine Serine Threonine Methionine Glycine

In specific embodiments, the number of substitutions, additions ordeletions in the amino acid sequence of FIGS. 1A-D and/or any of thepolypeptide fragments described herein (e.g., the extracellular domainor intracellular domain) is 75, 70, 60, 50, 40, 35, 30, 25, 20, 15, 10,9, 8, 7, 6, 5, 4, 3, 2, 1 or 30-20, 20-15, 20-10, 15-10, 10-1, 5-10,1-5, 1-3 or 1-2.

Amino acids in the TR9 protein of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding or in vitro proliferative activity.Sites that are critical for ligand-receptor binding can also bedetermined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith et al., J. Mol.Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).

The polypeptides of the present invention are preferably provided in anisolated form. By “isolated polypeptide” is intended a polypeptideremoved from its native environment. Thus, a polypeptide produced and/orcontained within a recombinant host cell is considered isolated forpurposes of the present invention. Also intended as an “isolatedpolypeptide” are polypeptides that have been purified, partially orsubstantially, from a recombinant host cell. For example, arecombinantly produced version of the TR9 receptor can be substantiallypurified by the one-step method described in Smith and Johnson, Gene67:31-40 (1988).

The polypeptides of the present invention include the polypeptideencoded by the deposited cDNA including the leader; the maturepolypeptide encoded by the deposited cDNA minus the leader (i.e., themature protein); a polypeptide comprising amino acids about −40 to about615 in SEQ ID NO:2; a polypeptide comprising amino acids about −39 toabout 615 in SEQ ID NO:2; a polypeptide comprising amino acids about 1to about 615 in SEQ ID NO:2; a polypeptide comprising the extracellulardomain; a polypeptide comprising the four TNFR-like cysteine rich motifsof TR9 (amino acid residues 67 to 211 in FIGS. 1A-D; amino acid residues27-171 in SEQ ID NO:2); a polypeptide comprising the transmembranedomain; a polypeptide comprising the intracellular domain; a polypeptidecomprising the extracellular and intracellular domains with all or partof the transmembrane domain deleted; a polypeptide comprising the deathdomain (amino acid residues 429-495 as depicted in FIGS. 1A-D; aminoacid residues 389-455 in SEQ ID NO:2); and/or a polypeptide comprisingthe TR9 leucine zipper (amino acid residues 497-518 of FIGS. 1A-D; aminoacid residues 457-478 of SEQ ID NO:2); as well as polypeptides which areat least 80% identical, more preferably at least 90% or 95% identical,still more preferably at least 96%, 97%, 98% or 99% identical to thepolypeptides described above, and also include portions of suchpolypeptides with at least 30 amino acids and more preferably at least50 amino acids.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of a TR9 receptorpolypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the TR9 receptor. In otherwords, to obtain a polypeptide having an amino acid sequence at least95% identical to a reference amino acid sequence, up to 5% of the aminoacid residues in the reference sequence may be deleted or substitutedwith another amino acid, or a number of amino acids up to 5% of thetotal amino acid residues in the reference sequence may be inserted intothe reference sequence. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequence shown in FIGS. 1A-D (SEQ ID NO:2), the amino acid sequenceencoded by deposited cDNA clone, or fragments thereof, can be determinedconventionally using known computer programs such the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). When using Bestfit or any other sequence alignment programto determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set, of course, such that the percentage of identityis calculated over the full length of the reference amino acid sequenceand that gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDBamino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1,Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter. According to this embodiment, if the subject sequence isshorter than the query sequence due to N— or C-terminal deletions, notbecause of internal deletions, a manual correction is made to theresults to take into consideration the fact that the FASTDB program doesnot account for N— and C-terminal truncations of the subject sequencewhen calculating global percent identity. For subject sequencestruncated at the N— and C-termini, relative to the query sequence, thepercent identity is corrected by calculating the number of residues ofthe query sequence that are N— and C-terminal of the subject sequence,which are not matched/aligned with a corresponding subject residue, as apercent of the total bases of the query sequence. A determination ofwhether a residue is matched/aligned is determined by results of theFASTDB sequence alignment. This percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thisfinal percent identity score is what is used for the purposes of thisembodiment. Only residues to the N— and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N— andC-terminal residues of the subject sequence. For example, a 90 aminoacid residue subject sequence is aligned with a 100 residue querysequence to determine percent identity. The deletion occurs at theN-terminus of the subject sequence and therefore, the FASTDB alignmentdoes not show a matching/alignment of the first 10 residues at theN-terminus. The 10 unpaired residues represent 10% of the sequence(number of residues at the N— and C-termini not matched/total number ofresidues in the query sequence) so 10% is subtracted from the percentidentity score calculated by the FASTDB program. If the remaining 90residues were perfectly matched the final percent identity would be 90%.In another example, a 90 residue subject sequence is compared with a 100residue query sequence. This time the deletions are internal deletionsso there are no residues at the N— or C-termini of the subject sequencewhich are not matched/aligned with the query. In this case the percentidentity calculated by FASTDB is not manually corrected. Once again,only residue positions outside the N— and C-terminal ends of the subjectsequence, as displayed in the FASTDB alignment, which are notmatched/aligned with the query sequence are manually corrected for. Noother manual corrections are made for the purposes of this embodiment.

The polypeptides of the present invention have uses which include, butare not limited to, molecular weight marker on SDS-PAGE gels or onmolecular sieve gel filtration columns using methods well known to thoseof skill in the art.

For many proteins, including the extracellular domain of a membraneassociated protein or the mature form(s) of a secreted protein, it isknown in the art that one or more amino acids may be deleted from theN-terminus or C-terminus without substantial loss of biologicalfunction. However, even if deletion of one or more amino acids from theN-terminus or C-terminus of a protein results in modification or loss ofone or more biological functions of the protein, other TR9 functionalactivities may still be retained. For example, in many instances, theability of the shortened protein to induce and/or bind to antibodieswhich recognize TR9 (preferably antibodies that bind specifically toTR9) will retained irrespective of the size or location of the deletion.Whether a particular polypeptide lacking N-terminal and/or C-terminalresidues of a complete protein retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art.

In one embodiment, the present invention further provides polypeptideshaving one or more residues deleted from the amino terminus of the aminoacid sequence of the TR9 polypeptide depicted in FIGS. 1A-D (SEQ IDNO:2) or encoded by the cDNA of the deposited clone. Particularly, inone embodiment, N-terminal deletions of the TR9 polypeptide can bedescribed by the general formula m to 615, where m is a number from −39to 614 corresponding to the position of amino acid identified in SEQ IDNO:2 and preferably, corresponds to one of the N-terminal amino acidresidues identified in the N-terminal deletions specified herein. Inspecific embodiments, N-terminal deletions of the TR9 polypeptide of theinvention comprise, or alternatively consist of, amino acid residues:Q-2 to L-615; P-3 to L-615; E-4 to L-615; Q-5 to L-615; K-6 to L-615;A-7 to L-615; S-8 to L-615; N-9 to L-615; L-10 to L-615; I-11 to L-615;G-12 to L-615; T-13 to L-615; Y-14 to L-615; R-15 to L-615; H-16 toL-615; V-17 to L-615; D-18 to L-615; R-19 to L-615; A-20 to L-615; T-21to L-615; G-22 to L-615; Q-23 to L-615; V-24 to L-615; L-25 to L-615;T-26 to L-615; C-27 to L-615; D-28 to L-615; K-29 to L-615; C-30 toL-615; P-31 to L-615; A-32 to L-615; G-33 to L-615; T-34 to L-615; Y-35to L-615; V-36 to L-615; S-37 to L-615; E-38 to L-615; H-39 to L-615;C-40 to L-615; T-41 to L-615; N-42 to L-615; T-43 to L-615; S-44 toL-615; L-45 to L-615; R-46 to L-615; V-47 to L-615; C-48 to L-615; S-49to L-615; S-50 to L-615; C-51 to L-615; P-52 to L-615; V-53 to L-615;G-54 to L-615; T-55 to L-615; F-56 to L-615; T-57 to L-615; R-58 toL-615; H-59 to L-615; E-60 to L-615; N-61 to L-615; G-62 to L-615; I-63to L-615; E-64 to L-615; K-65 to L-615; C-66 to L-615; H-67 to L-615;D-68 to L-615; C-69 to L-615; S-70 to L-615; Q-71 to L-615; P-72 toL-615; C-73 to L-615; P-74 to L-615; W-75 to L-615; P-76 to L-615; M-77to L-615; I-78 to L-615; E-79 to L-615; K-80 to L-615; L-81 to L-615;P-82 to L-615; C-83 to L-615; A-84 to L-615; A-85 to L-615; L-86 toL-615; T-87 to L-615; D-88 to L-615; R-89 to L-615; E-90 to L-615; C-91to L-615; T-92 to L-615; C-93 to L-615; P-94 to L-615; P-95 to L-615;G-96 to L-615; M-97 to L-615; F-98 to L-615; Q-99 to L-615; S-100 toL-615; N-101 to L-615; A-102 to L-615; T-103 to L-615; C-104 to L-615;A-105 to L-615; P-106 to L-615; H-107 to L-615; T-108 to L-615; V-109 toL-615; C-110 to L-615; P-111 to L-615; V-112 to L-615; G-113 to L-615;W-114 to L-615; G-115 to L-615; V-116 to L-615; R-117 to L-615; K-118 toL-615; K-119 to L-615; G-120 to L-615; T-121 to L-615; E-122 to L-615;T-123 to L-615; E-124 to L-615; D-125 to L-615; V-126 to L-615; R-127 toL-615; C-128 to L-615; K-129 to L-615; Q-130 to L-615; C-131 to L-615;A-132 to L-615; R-133 to L-615; G-134 to L-615; T-135 to L-615; F-136 toL-615; S-137 to L-615; D-138 to L-615; V-139 to L-615; P-140 to L-615;S-141 to L-615; S-142 to L-615; V-143 to L-615; M-144 to L-615; K-145 toL-615; C-146 to L-615; K-147 to L-165; A-148 to L-615; Y-149 to L-615;T-150 to L-615; D-151 to L-615; C-152 to L-615; L-153 to L-615; S-154 toL-615; Q-155 to L-615; N-156 to L-615; L-157 to L-615; V-158 to L-615;V-159 to L-615; I-60 to L-615; K-161 to L-615; P-162 to L-615; G-163 toL-615; T-164 to L-615; K-165 to L-615; E-166 to L-615; T-167 to L-615;D-168 to L-615; N-169 to L-615; V-170 to L-615; C-171 to L-615; G-172 toL-615; T-173 to L-615; L-174 to L-615; P-175 to L-615; S-176 to L-615;F-177 to L-615; S-178 to L-615; S-179 to L-615; S-180 to L-615; T-181 toL-615; S-182 to L-615; P-183 to L-615; S-184 to L-615; P-185 to L-615;G-186 to L-615; T-187 to L-615; A-188 to L-615; I-189 to L-615; F-190 toL-615; P-191 to L-615; R-192 to L-615; P-193 to L-615; E-194 to L-615;H-195 to L-615; M-196 to L-615; E-197 to L-615; T-198 to L-615; H-199 toL-615; E-200 to L-615; V-201 to L-615; P-202 to L-615; S-203 to L-615;S-204 to L-615; T-205 to L-615; Y-206 to L-615; V-207 to L-615; P-208 toL-615; K-209 to L-615; G-210 to L-615; M-211 to L-615; N-212 to L-615;S-213 to L-615; T-214 to L-615; E-215 to L-615; S-216 to L-615; N-217 toL-615; S-218 to L-615; S-219 to L-615; A-220 to L-615; S-221 to L-615;V-222 to L-615; R-223 to L-615; P-224 to L-615; K-225 to L-615; V-226 toL-615; L-227 to L-615; S-228 to L-615; S-229 to L-615; I-230 to L-615;Q-231 to L-615; E-232 to L-615; G-233 to L-615; T-234 to L-615; V-235 toL-615; P-236 to L-615; D-237 to L-615; N-238 to L-615; T-239 to L-615;S-240 to L-615; S-241 to L-615; A-242 to L-615; R-243 to L-615; G-244 toL-615; K-245 to L-615; E-246 to L-615; D-247 to L-615; V-248 to L-615;N-249 to L-615; K-250 to L-615; T-251 to L-615; L-252 to L-615; P-253 toL-615; N-254 to L-615; L-255 to L-615; Q-256 to L-615; V-257 to L-615;V-258 to L-615; N-259 to L-615; H-260 to L-615; Q-261 to L-615; Q-262 toL-615; G-263 to L-615; P-264 to L-615; H-265 to L-615; H-266 to L-615;R-267 to L-615; H-268 to L-615; I-269 to L-615; L-270 to L-615; K-271 toL-615; L-272 to L-615; L-273 to L-615; P-274 to L-615; S-275 to L-615;M-276 to L-615; E-277 to L-615; A-278 to L-615; T-279 to L-615; G-280 toL-615; G-281 to L-615; E-282 to L-615; K-283 to L-615; S-284 to L-615;S-285 to L-615; T-286 to L-615; P-287 to L-615; I-288 to L-615; K-289 toL-615; G-290 to L-615; P-291 to L-615; K-292 to L-615; R-293 to L-615;G-294 to L-615; H-295 to L-615; P-296 to L-615; R-297 to L-615; Q-298 toL-615; N-299 to L-615; L-300 to L-615; H-301 to L-615; K-302 to L-615;H-303 to L-615; F-304 to L-615; D-305 to L-615; I-306 to L-615; N-307 toL-615; E-308 to L-615; H-309 to L-615; L-310 to L-615; P-311 to L-615;W-312 to L-615; M-313 to L-615; I-314 to L-615; V-315 to L-615; L-316 toL-615; F-317 to L-615; L-318 to L-615; L-319 to L-615; L-320 to L-615;V-321 to L-615; L-322 to L-615; V-323 to L-615; V-324 to L-615; I-325 toL-615; V-326 to L-615; V-327 to L-615; C-328 to L-615; S-329 to L-615;I-330 to L-615; R-331 to L-615; K-332 to L-615; S-333 to L-615; S-334 toL-615; R-335 to L-615; T-336 to L-615; L-337 to L-615; K-338 to L-615;K-339 to L-615; G-340 to L-615; P-341 to L-615; R-342 to L-615; Q-343 toL-615; D-344 to L-615; P-345 to L-615; S-346 to L-615; A-347 to L-615;I-348 to L-615; V-349 to L-615; E-350 to L-615; K-351 to L-615; A-352 toL-615; G-353 to L-615; L-354 to L-615; K-355 to L-615; K-356 to L-615;S-357 to L-615; M-358 to L-615; T-359 to L-615; P-360 to L-615; T-361 toL-615; Q-362 to L-615; N-363 to L-615; R-364 to L-615; E-365 to L-615;K-366 to L-615; W-367 to L-615; I-368 to L-615; Y-369 to L-615; Y-370 toL-615; C-371 to L-615; N-372 to L-615; G-373 to L-615; H-374 to L-615;G-375 to L-615; I-376 to L-615; D-377 to L-615; I-378 to L-615; L-379 toL-615; K-380 to L-615; L-381 to L-615; V-382 to L-615; A-383 to L-615;A-384 to L-615; Q-385 to L-615; V-386 to L-615; G-387 to L-615; S-388 toL-615; Q-389 to L-615; W-390 to L-615; K-391 to L-615; D-392 to L-615;I-393 to L-615; Y-394 to L-615; Q-395 to L-615; F-396 to L-615; L-397 toL-615; C-398 to L-615; N-399 to L-615; A-400 to L-615; S-401 to L-615;E-402 to L-615; R-403 to L-615; E-404 to L-615; V-405 to L-615; A-406 toL-615; A-407 to L-615; F-408 to L-615; S-409 to L-615; N-410 to L-615;G-411 to L-615; Y-412 to L-615; T-413 to L-615; A-414 to L-615; D-415 toL-615; H-416 to L-615; E-417 to L-615; R-418 to L-615; A-419 to L-615;Y-420 to L-615; A-421 to L-615; A-422 to L-615; L-423 to L-615; Q-424 toL-615; H-425 to L-615; W-426 to L-615; T-427 to L-615; I-428 to L-615;R-429 to L-615; G-430 to L-615; P-431 to L-615; E-432 to L-615; A-433 toL-615; S-434 to L-615; L-435 to L-615; A-435 to L-615; Q-437 to L-615;L-438 to L-615; I-439 to L-615; S-440 to L-615; A-441 to L-615; L-442 toL-615; R-443 to L-615; Q-444 to L-615; H-445 to L-615; R-446 to L-615;R-447 to L-615; N-448 to L-615; D-449 to L-615; V-450 to L-615; V-451 toL-615; E-452 to L-615; K-453 to L-615; I-454 to L-615; R-455 to L-615;G-456 to L-615; L-457 to L-615; M-458 to L-615; E-459 to L-615; D-460 toL-615; T-461 to L-615; T-462 to L-615; Q-463 to L-615; L-464 to L-615;E-465 to L-615; T-466 to L-615; D-467 to L-615; K-468 to L-615; L-469 toL-615; A-470 to L-615; L-471 to L-615; P-472 to L-615; M-473 to L-615;S-474 to L-615; P-475 to L-615; S-476 to L-615; P-477 to L-615; L-478 toL-615; S-479 to L-615; P-480 to L-615; S-481 to L-615; P-482 to L-615;I-483 to L-615; P-484 to L-615; S-485 to L-615; P-486 to L-615; N-487 toL-615; A-488 to L-615; K-489 to L-615; L-490 to L-615; E-491 to L-615;N-492 to L-615; S-493 to L-615; A-494 to L-615; L-495 to L-615; L-496 toL-615; T-497 to L-615; V-498 to L-615; E-499 to L-615; P-500 to L-615;S-501 to L-615; P-502 to L-615; Q-503 to L-615; D-504 to L-615; K-505 toL-615; N-506 to L-615; K-507 to L-615; G-508 to L-615; F-509 to L-615;F-510 to L-615; V-511 to L-615; D-512 to L-615; E-513 to L-615; S-514 toL-615; E-515 to L-615; P-516 to L-615; L-517 to L-615; L-518 to L-615;R-519 to L-615; C-520 to L-615; D-521 to L-615; S-522 to L-615; T-523 toL-615; S-524 to L-615; S-525 to L-615; G-526 to L-615; S-527 to L-615;S-528 to L-615; A-529 to L-615; L-530 to L-615; S-531 to L-615; R-532 toL-615; N-533 to L-615; G-534 to L-615; S-535 to L-615; F-536 to L-615;I-537 to L-615; T-538 to L-615; K-539 to L-615; E-540 to L-615; K-541 toL-615; K-542 to L-615; D-543 to L-615; T-544 to L-615; V-545 to L-615;L-546 to L-615; R-547 to L-615; Q-548 to L-615; V-549 to L-615; R-550 toL-615; L-551 to L-615; D-552 to L-615; P-553 to L-615; C-554 to L-615;D-555 to L-615; L-556 to L-615; Q-557 to L-615; P-558 to L-615; I-559 toL-615; F-560 to L-615; D-561 to L-615; D-562 to L-615; M-563 to L-615;L-564 to L-615; H-565 to L-615; F-566 to L-615; L-567 to L-615; N-568 toL-615; P-569 to L-615; E-570 to L-615; E-571 to L-615; L-572 to L-615;R-573 to L-615; V-574 to L-615; I-575 to L-615; E-576 to L-615; E-577 toL-615; I-578 to L-615; P-579 to L-615; Q-580 to L-615; A-581 to L-615;E-582 to L-615; D-583 to L-615; K-584 to L-615; L-585 to L-615; D-586 toL-615; R-587 to L-615; L-588 to L-615; F-589 to L-615; E-590 to L-615;I-591 to L-615; I-592 to L-615; G-593 to L-615; V-594 to L-615; K-595 toL-615; S-596 to L-615; Q-597 to L-615; E-598 to L-615; A-599 to L-615;S-600 to L-615; Q-601 to L-615; T-602 to L-615; L-603 to L-615; L-604 toL-615; D-605 to L-615; S-606 to L-615; V-607 to L-615; Y-608 to L-615;S-609 to L-615; H-610 to L-615; of SEQ ID NO:2. Polynucleotides encodingthese polypeptides are also encompassed by the invention.

In another embodiment, N-terminal deletions of the TR9 polypeptide canbe described by the general formula m to 310 where m is a number from−40 to 309 corresponding to the amino acid sequence identified in SEQ IDNO:2. In specific embodiments, N terminal deletions of the TR9 of theinvention comprise, or alternatively, consist of, amino acid residues:Q-2 to L-310; P-3 to L-310; E-4 to L-310; Q-5 to L-310; K-6 to L-310;A-7 to L-310; S-8 to L-310; N-9 to L-310; L-10 to L-310; I-11 to L-310;G-12 to L-310; T-13 to L-310; Y-14 to L-310; R-15 to L-310; H-16 toL-310; V-17 to L-310; D-18 to L-310 R-19 to L-310; A-20 to L-310; T-21to L-310; G-22 to L-310; Q-23 to L-310; V-24 to L-310; L-25 to L-310;T-26 to L-310; C-27 to L-310; D-28 to L-310; K-29 to L-310; C-30 toL-310; P-31 to L-310; A-32 to L-310; G-33 to L-310; T-34 to L-310; Y-35to L-310; V-36 to L-310; S-37 to L-310; E-38 to L-310; H-39 to L-310;C-40 to L-310; T-41 to L-310; N-42 to L-310; T-43 to L-310; S-44 toL-310; L-45 to L-310; R-46 to L-310; V-47 to L-310; C-48 to L-310; S-49to L-310; S-50 to L-310; C-51 to L-310; P-52 to L-310; V-53 to L-310;G-54 to L-310; T-55 to L-310; F-56 to L-310; T-57 to L-310; R-58 toL-310; H-59 to L-310; E-60 to L-310; N-61 to L-310; G-62 to L-310; I-63to L-310; E-64 to L-310; K-65 to L-310; C-66 to L-310; H-67 to L-310;D-68 to L-310; C-69 to L-310; S-70 to L-310; Q-71 to L-310; P-72 toL-310; C-73 to L-310; P-74 to L-310; W-75 to L-310; P-76 to L-310; M-77to L-310; I-78 to L-310; E-79 to L-310; K-80 to L-310; L-81 to L-310;P-82 to L-310; C-83 to L-310; A-84 to L-310; A-85 to L-310; L-86 toL-310; T-87 to L-310; D-88 to L-310; R-89 to L-310; E-90 to L-310; C-91to L-310; T-92 to L-310; C-93 to L-310; P-94 to L-310; P-95 to L-310;G-96 to L-310; M-97 to L-310; F-98 to L-310; Q-99 to L-310; S-100 toL-310; N-101 to L-310; A-102 to L-310; T-103 to L-310; C-104 to L-310;A-105 to L-310; P-106 to L-310; H-107 to L-310; T-108 to L-310; V-109 toL-310; C-110 to L-310; P-111 to L-310; V-112 to L-310; G-113 to L-310;W-114 to L-310; G-115 to L-310; V-116 to L-310; R-117 to L-310; K-118 toL-310; K-119 to L-310; G-120 to L-310; T-121 to L-310; E-122 to L-310;T-123 to L-310; E-124 to L-310; D-125 to L-310; V-126 to L-310; R-127 toL-310; C-128 to L-310; K-129 to L-310; Q-130 to L-310; C-131 to L-310;A-132 to L-310; R-133 to L-310; G-134 to L-310; T-135 to L-310; F-136 toL-310; S-137 to L-310; D-138 to L-310; V-139 to L-310; P-140 to L-310;S-141 to L-310; S-142 to L-310; V-143 to L-310; M-144 to L-310; K-145 toL-310; C-146 to L-310; K-147 to L-310; A-148 to L-310; Y-149 to L-310;T-150 to L-310; D-151 to L-310; C-152 to L-310; L-153 to L-310; S-154 toL-310; Q-155 to L-310; N-156 to L-310; L-157 to L-310; V-158 to L-310;V-159 to L-310; I-160 to L-310; K-161 to L-310; P-162 to L-310; G-163 toL-310; T-164 to L-310; K-165 to L-310; E-166 to L-310; T-167 to L-310;D-168 to L-310; N-169 to L-310; V-170 to L-310; C-171 to L-310; G-172 toL-310; T-173 to L-310; L-174 to L-310; P-175 to L-310; S-176 to L-310;F-177 to L-310; S-178 to L-310; S-179 to L-310; S-180 to L-310; T-181 toL-310; S-182 to L-310; P-183 to L-310; S-184 to L-310; P-185 to L-310;G-186 to L-310; T-187 to L-310; A-188 to L-310; I-189 to L-310; F-190 toL-310; P-191 to L-310; R-192 to L-310 P-193 to L-310; E-194 to L-310;H-195 to L-310; M-196 to L-310; E-197 to L-310; T-198 to L-310; H-199 toL-310; E-200 to L-310; V-201 to L-310; P-202 to L-310; S-203 to L-310;S-204 to L-310; T-205 to L-310; Y-206 to L-310; V-207 to L-310; P-208 toL-310; K-209 to L-310; G-210 to L-310; M-211 to L-310; N-212 to L-310;S-213 to L-310; T-214 to L-310; E-215 to L-310; S-216 to L-310; N-127 toL-310; S-218 to L-310; S-219 to L-310; A-220 to L-310; S-221 to L-310;V-222 to L-310; R-223 to L-310; P-224 to L-310; K-225 to L-310; V-226 toL-310; L-227 to L-310; S-228 to L-310; S-229 to L-310; I-230 to L-310;Q-231 to L-310; E-232 to L-310; G-233 to L-310; T-234 to L-310; V-235 toL-310; P-236 to L-310; D-237 to L-310; N-238 to L-310; T-239 to L-310;S-240 to L-310; S-241 to L-310; A-242 to L-310; R-243 to L-310; G-244 toL-310; K-245 to L-310; E-246 to L-310; D-247 to L-310; V-248 to L-310;N-249 to L-310; K-250 to L-310; T-251 to L-310; L-252 to L-310; P-253 toL-310; N-254 to L-310; L-255 to L-310; Q-256 to L-310; V-257 to L-310;V-258 to L-310; N-259 to L-310; H-260 to L-310; Q-261 to L-310; Q-262 toL-310; G-263 to L-310; P-264 to L-310; H-265 to L-310; H-266 to L-310;R-267 to L-310; H-268 to L-310; I-269 to L-310; L-270 to L-310; K-271 toL-310; L-272 to L-310; L-273 to L-310; P-274 to L-310; S-275 to L-310;M-276 to L-310; E-277 to L-310; A-278 to L-310; T-279 to L-310; G-280 toL-310; G-281 to L-310; E-282 to L-310; K-283 to L-310; S-284 to L-310;S-285 to L-310; T-286 to L-310; P-287 to L-310; I-288 to L-310; K-289 toL-310; G-290 to L-310; P-291 to L-310; K-292 to L-310; R-293 to L-310;G-294 to L-310; H-295 to L-310; P-296 to L-310; R-297 to L-310; Q-298 toL-310; N-299 to L-310; L-300 to L-310; H-301 to L-310; K-302 to L-310;H-303 to L-310; F-304 to L-310; D-305 to L-310; of SEQ ID NO:2.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

Further embodiments of the invention are directed to C-terminaldeletions of the TR9 polypeptide described by the general formula 1 ton, where n is a number from 2 to 614 corresponding to the position ofamino acid residue identified in SEQ ID NO:2 and preferably, correspondsto one of the C-terminal amino acid residues identified in theC-terminal deletions specified herein. In specific embodiments, Cterminal deletions of the TR9 polypeptide of the invention comprise, oralternatively, consist of, amino acid residues: A-1 to L-614; A-1 toD-613; A-1 to P-612; A-1 to L-611; A-1 to H-610; A-1 to S-609; A-1 toY-608; A-1 to V-607; A-1 to S-606; A-1 to D-605; A-1 to L-604; A-1 toL-603; A-1 to T-602; A-1 to Q-601; A-1 to S-600; A-1 to A-599; A-1 toE-598; A-1 to Q-597; A-1 to S-596; A-1 to K-595; A-1 to V-594; A-1 toG-593; A-1 to I-592; A-1 to I-591; A-1 to E-590; A-1 to F-589; A-1 toL-588; A-1 to R-587; A-1 to D-586; A-1 to L-585; A-1 to K-584; A-1 toD-583; A-1 to E-582; A-1 to A-581; A-1 to Q-580; A-1 to P-579; A-1 toI-578; A-1 to E-577; A-1 to E-576; A-1 to I-575; A-1 to V-574; A-1 toR-573; A-1 to L-572; A-1 to E-571; A-1 to E-570; A-1 to P-569; A-1 toN-568; A-1 to L-567; A-1 to F-566; A-1 to H-565; A-1 to L-564; A-1 toM-563; A1 to D-562; A-1 to D-561; A-1 to F-560; A-1 to I-559; A-1 toP-558; A-1 to Q-557; A-1 to L-556; A-1 to D-555; A-1 to C-554; A-1 toP-553; A-1 to D-552; A-1 to L-551; A-1 to R-550; A-1 to V-549; A-1 toQ-548; A-1 to R-547; A-1 to L-546; A-1 to V-545; A-1 to T-544; A-1 toD-543; A-1 to K-542; A-1 to K-541; A-1 to E-540; A-1 to K-539; A-1 toT-538; A-1 to I-537; A-1 to F-536; A-1 to S-535; A-1 to G-534; A-1 toN-533; A-1 to R-532; A-1 to S-531; A-1 to L-530; A-1 to A-529; A-1 toS-528; A-1 to S-527; A-1 to G-526; A-1 to S-525; A-1 to S-524; A-1 toT-523; A-1 to S-522; A-1 to D-521; A-1 to C-520; A-1 to R-519; A-1 toL-518; A-1 to L-517; A-1 to P-516; A-1 to E-515; A-1 to S-514; A-1 toE-513; A-1 to D-512; A-1 to V-511; A-1 to F-510; A-1 to F-509; A-1 toG-508; A-1 to K-507; A-1 to N-506; A-1 to K-505; A-1 to D-504; A-1 toQ-503; A-1 to P-502; A-1 to S-501; A-1 to P-500; A-1 to E-499; A-1 toV-498; A-1 to T-497; A-1 to L-496; A-1 to L-495; A-1 to A-494; A-1 toS-493; A-1 to N-492; A-1 to E-491; A-1 to L-490; A-1 to K-489; A-1 toA-488; A-1 to N-487; A-1 to P-486; A-1 to S-485; A-1 to P-484; A-1 toI-483; A-1 to P-482; A-1 to S-481; A-1 to P-480; A-1 to S-479; A-1 toL-478; A-1 to P-477; A-1 to S-476; A-1 to P-475; A-1 to S-474; A-1 toM-473; A-1 to P-472; A-1 to L-471; A-1 to A-470; A-1 to L-469; A-1 toK-468; A-1 to D-467; A-1 to T-466; A-1 to E-465; A-1 to L-464; A-1 toQ-463; A-1 to T-462; A-1 to T-461; A-1 to D-460; A-1 to E-459; A-1 toM-458; A-1 to L-457; A-1 to G-456; A-1 to R-455; A-1 to I-454; A-1 toK-453; A-1 to E-452; A-1 to V-451; A-1 to V-450; A-1 to D-449; A-1 toN-448; A-1 to R-447; A-1 to R-446; A-1 to H-445; A-1 to Q-444; A-1 toR-443; A-1 to L-442; A-1 to A-441; A-1 to S-440; A-1 to I-439; A-1 toL-438; A-1 to Q-437; A-1 to A-436; A-1 to L-435; A-1 to S-434; A-1 toA-433; A-1 to E-432; A-1 to P-431; A-1 to G-430; A-1 to R-429; A-1 toI-428; A-1 to T-427; A-1 to W-426; A-1 to H-425; A-1 to Q-424; A-1 toL-423; A-1 to A-422; A-1 to A-421; A-1 to Y-420; A-1 to A-419; A-1 toR-418; A-1 to E-417; A-1 to H-416; A-1 to D-415; A-1 to A-414; A-1 toT-413; A-1 to Y-412; A-1 to G-411; A-1 to N-410; A-1 to S-409; A-1 toF-408; A-1 to A-407; A-1 to A-406; A-1 to V-405; A-1 to E-404; A-1 toR-403; A-1 to E-402; A-1 to S-401; A-1 to A-400; A-1 to N-399; A-1 toC-398; A-1 to L-397; A-1 to F-396; A-1 to Q-395; A-1 to Y-394; A-1 toI-393; A-1 to D-392; A-1 to K-391; A-1 to W-390; A-1 to Q-389; A-1 toS-388; A-1 to G-387; A-1 to V-386; A-1 to Q-385; A-1 to A-384; A-1 toA-383; A-1 to V-382; A-1 to L-381; A-1 to K-380; A-1 to L-379; A-1 toI-378; A-1 to D-377; A-1 to I-376; A-1 to G-375; A-1 to H-374; A-1 toG-373; A-1 to N-372; A-1 to C-371; A-1 to Y-370; A-1 to Y-369; A-1 toI-368; A-1 to W-367; A-1 to K-366; A-1 to E-365; A-1 to R-364; A-1 toN-363; A-1 to Q-362; A-1 to T-361; A-1 to P-360; A-1 to T-359; A-1 toM-358; A-1 to S-357; A-1 to K-356; A-1 to K-355; A-1 to L-354; A-1 toG-353; A-1 to A-352; A-1 to K-351; A-1 to E-350; A-1 to V-349; A-1 toI-348; A-1 to A-347; A-1 to S-346; A-1 to P-345; A-1 to D-344; A-1 toQ-343; A-1 to R-342; A-1 to P-341; A-1 to G-340; A-1 to K-339; A-1 toK-338; A-1 to L-337; A-1 to T-336; A-1 to R-335; A-1 to S-334; A-1 toS-333; A-1 to K-332; A-1 to R-331; A-1 to I-330; A-1 to S-329; A-1 toC-328; A-1 to V-327; A-1 to V-326; A-1 to I-325; A-1 to V-324; A-1 toV-323; A-1 to L-322; A-1 to V-321; A-1 to L-320; A-1 to L-319; A-1 toL-318; A-1 to F-317; A-1 to L-316; A-1 to V-315; A-1 to I-314; A-1 toM-313; A-1 to W-312; A-1 to P-311; A-1 to L-310; A-1 to H-309; A-1 toE-308; A-1 to N-307; A-1 to I-306; A-1 to D-305; A-1 to F-304; A-1 toH-303; A-1 to K-302; A-1 to H-301; A-1 to L-300; A-1 to N-299; A-1 toQ-298; A-1 to R-297; A-1 to P-296; A-1 to H-295; A-1 to G-294; A-1 toR-293; A-1 to K-292; A-1 to P-291; A-1 to G-290; A-1 to K-289; A-1 toI-288; A-1 to P-287; A-1 to T-286; A-1 to S-285; A-1 to S-284; A-1 toK-283; A-1 to E-282; A-1 to G-281; A-1 to G-280; A-1 to T-279; A-1 toA-278; A-1 to E-277; A-1 to M-276; A-1 to S-275; A-1 to P-274; A-1 toL-273; A-1 to L-272; A-1 to K-271; A-1 to L-270; A-1 to I-269; A-1 toH-268; A-1 to R-267; A-1 to H-266; A-1 to H-265; A-1 to P-264; A-1 toG-263; A-1 to Q-262; A-1 to Q-261; A-1 to H-260; A-1 to N-259; A-1 toV-258; A-1 to V-257; A-1 to Q-256; A-1 to L-255; A-1 to N-254; A-1 toP-253; A-1 to L-252; A-1 to T-251; A-1 to K-250; A-1 to N-249; A-1 toV-248; A-1 to D-247; A-1 to E-246; A-1 to K-245; A-1 to G-244; A-1 toR-243; A-1 to A-242; A-1 to S-241; A-1 to S-240; A-1 to T-239; A-1 toN-238; A-1 to D-237; A-1 to P-236; A-1 to V-235; A-1 to T-234; A-1 toG-233; A-1 to E-232; A-1 to Q-231; A-1 to I-230; A-1 to S-229; A-1 toS-228; A-1 to L-227; A-1 to V-226; A-1 to K-225; A-1 to P-224; A-1 toR-223; A-1 to V-222; A-1 to S-221; A-1 to A-220; A-1 to S-219; A-1 toS-218; A-1 to N-217; A-1 to S-216; A-1 to E-215; A-1 to T-214; A-1 toS-213; A-1 to N-212; A-1 to M-211; A-1 to G-210; A-1 to K-209; A-1 toP-208; A-1 to V-207; A-1 to Y-206; A-1 to T-205; A-1 to S-204; A-1 toS-203; A-1 to P-202; A-1 to V-201; A-1 to E-200; A-1 top H-199; A-1 toT-198; A-1 to E-197; A-1 to M-196; A-1 to H-195; A-1 to E-194; A-1 toP-193; A-1 to R-192; A-1 to P-191; A-1 to F-190; A-1 to I-189; A-1 toA-188; A-1 to T-187; A-1 to G-186; A-1 to P-185; A-1 to S-184; A-1 toP-183; A-1 to S-182; A-1 to T-181; A-1 to S-180; A-1 to S-179; A-1 toS-178; A-1 to F-177; A-1 to S-176; A-1 to P-175; A-1 to L-174; A-1 toT-173; A-1 to G-172; A-1 to C-171; A-1 to V-170; A-1 to N-169; A-1 toD-168; A-1 to T-167; A-1 to E-166; A-1 to K-165; A-1 to T-164; A-1 toG-163; A-1 to P-162; A-1 to K-161; A-1 to I-160; A-1 to V-159; A-1 toV-158; A-1 to L-157; A-1 to N-156; A-1 to Q-155; A-1 to S-154; A-1 toL-153; A-1 to C-152; A-1 to D-151; A-1 to T-150; A-1 to Y-149; A-1 toA-148; A-1 to K-147; A-1 to C-146; A-1 to K-145; A-1 to M-144; A-1 toV-143; A-1 to S-142; A-1 to S-141; A-1 to P-140; A-1 to V-139; A-1 toD-138; A-1 to S-137; A-1 to F-136; A-1 to T-135; A-1 to G-134; A-1 toR-133; A-1 to A-132; A-1 to C-131; A-1 to Q-130; A-1 to K-129; A-1 toC-128; A-1 to R-127; A-1 to V-126; A-1 to D-125; A-1 to E-124; A-1 toT-123; A-1 to E-122; A-1 to T-121; A-1 to G-120; A-1 to K-119; A-1 toK-118; A-1 to R-117; A-1 to V-116; A-1 to G-115; A-1 to W-114; A-1 toG-113; A-1 to V-112; A-1 to P-111; A-1 to C-110; A-1 to V-109; A-1 toT-108; A-1 to H-107; A-1 to P-106; A-1 to A-105; A-1 to C-104; A-1 toT-103; A-1 to A-102; A-1 to N-101; A-1 to S-100; A-1 to Q-99; A-1 toF-98; A-1 to M-97; A-1 to G-96; A-1 to P-95; A-1 to P-94; A-1 to C-93;A-1 to T-92; A-1 to C-91; A-1 to -90; A-1 to R-89; A-1 to D-88; A-1 toT-87; A-1 to L-86; A-1 to A-85; A-1 to A-84; A-1 to C-83; A-1 to P-82;A-1 to L-81; A-1 to K-80; A-1 to E-79; A-1 to I-78; A-1 to M-77; A-1 toP-76; A-1 to W-75; A-1 to P-74; A-1 to C-73; A-1 to P-72; A-1 to Q-71;A-1 to S-70; A-1 to C-69; A-1 to D-68; A-1 to H-67; A-1 to C-66; A-1 toK-65; A-1 to E-64; A-1 to I-63; A-1 to G-62; A-1 to N-61; A-1 to E-60;A-1 to H-59; A-1 to R-58; A-1 to T-57; A-1 to F-56; A-1 to T-55; A-1 toG-54; A-1 to V-53; A-1 to P-52; A-1 to C-51; A-1 to S-50; A-1 to S-49;A-1 to C-48; A-1 to V-47; A-1 to R-46; A-1 to L-45; A-1 to S-44; A-1 toT-43; A-1 to N-42; A-1 to T-41; A-1 to C-40; A-1 to H-39; A-1 to E-38;A-1 to S-37; A-1 to V-36; A-1 to Y-35; A-1 to T-34; A-1 to G-33; A-1 toA-32; A-1 to P-31; A-1 to C-30; A-1 to K-29; A-1 to D-28; A-1 to C-27;A-1 to T-26; A-1 to L-25; A-1 to V-24; A-1 to Q-23; A-1 to G-22; A-1 toT-21; A-1 to A-20; A-1 to R-19; A-1 to D-18; A-1 to V-17; A-1 to H-16;A-1 to R-15; A-1 to Y-14; A-1 to T-13; A-1 to G-12; A-1 to I-11; A-1 toL-10; A-1 to N-9; A-1 to S-8; A-1 to A-7; A-1 to K-6; of SEQ ID NO:2.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

Further embodiments of the invention are directed to polypeptidefragments comprising, or alternatively, consisting of, amino acidsdescribed by the general formula m to n, where m and n correspond to anyone of the amino acid residues specified above for these symbols,respectively. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

Polypeptide fragments of the present invention include polypeptidescomprising an amino acid sequence contained in SEQ ID NO:2, encoded bythe cDNA contained in the deposited clone, or encoded by nucleic acidswhich hybridize (e.g., under stringent hybridization conditions) to thenucleotide sequence contained in the deposited clone, or shown in FIGS.1A-D (SEQ ID NO:1) or the complementary strand thereto. Proteinfragments may be “free-standing,” or comprised within a largerpolypeptide of which the fragment forms a part or region, mostpreferably as a single continuous region. Representative examples ofpolypeptide fragments of the invention, include, for example, fragmentsthat comprise or alternatively, consist of, from about amino acidresidues −40 to 1, 1 to 20, 21 to 40, 41 to 60, 61 to 83, 84 to 100, 101to 120, 121 to 140, 141 to 160, 160-167, 161 to 180, 181 to 200, 201 to220, 221 to 240, 241 to 260, 261 to 280, 281 to 310, 311 to 350, 351 to400, 401 to 450, 451 to 500, 551 to 600, or 601 to the end of the codingregion of SEQ ID NO:2. Moreover, polypeptide fragments can be at leastabout 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150amino acids in length. In this context “about” includes the particularlyrecited ranges, larger or smaller by several (5, 4, 3, 2, or 1) aminoacids, at either extreme or at both extremes.

Among the especially preferred fragments of the invention are fragmentscharacterized by structural or functional attributes of TR9. Suchfragments include amino acid residues that comprise alpha-helix andalpha-helix forming regions (“alpha-regions”), beta-sheet andbeta-sheet-forming regions (“beta-regions”), turn and turn-formingregions (“turn-regions”), coil and coil-forming regions(“coil-regions”), hydrophillic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, surface forming regions,and high antigenic index regions (i.e., regions of polypeptidesconsisting of amino acid residues having an antigenic index of or equalto greater than 1.5, as identified using the default parameters of theJameson-Wolf program) of TR9. Certain preferred regions are thosedisclosed in FIG. 3 and include, but are not limited to, regions of theaforementioned types identified by analysis of the amino acid sequencedepicted in FIGS. 1A-D, such preferred regions include; Garnier-Robsonpredicted alpha-regions, beta-regions, turn-regions, and coil-regions;Chou-Fasman predicted alpha-regions, beta-regions, turn-regions, andcoil-regions; Kyte-Doolittle predicted hydrophilic and hydrophobicregions; Eisenberg alpha and beta amphipathic regions; Eminisurface-forming regions; and Jameson-Wolf high antigenic index regions,as predicted using the default parameters of these computer programs.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

In specific embodiments, polypeptide fragments of the inventioncomprise, or alternatively, consist of, amino acid residues: 40 to 48,40 to 51, 51 to 66, 66 to 73, 73 to 83, 83 to 104, 104 to 110, 110 to128, 128 to 146, 146 to 152, 40 to 152, and/or 28 to 171 in SEQ ID NO:2.

In other embodiments, the fragments or polypeptides of the invention(i.e., those described herein) are not larger than 610, 600, 580, 570,550, 525, 500, 475, 450, 400, 425, 390, 380, 375, 350, 336, 334, 331,305, 300, 295, 290, 285, 280, 275, 260, 250, 225, 200, 185, 175, 170,165, 160, 155, 150, 145, 140, 135, 130, 125, 120, 115, 110, 105, 100,90, 80, 75, 60, 50, 40, 30, or 25 amino acid residues in length.

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide of the invention.The epitope of this polypeptide portion is an immunogenic or antigenicepitope of a polypeptide described herein. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response whenthe whole protein is the immunogen. On the other hand, a region of aprotein molecule to which an antibody can bind is defined as an“antigenic epitope.” The number of immunogenic epitopes of a proteingenerally is less than the number of antigenic epitopes. See, forinstance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, J. G. Sutcliffe et al., “AntibodiesThat React With Predetermined Sites on Proteins,” Science 219:660-666(1983). Peptides capable of eliciting protein-reactive sera arefrequently represented in the primary sequence of a protein, can becharacterized by a set of simple chemical rules, and are confinedneither to immunodominant regions of intact proteins (i.e., immunogenicepitopes) nor to the amino or carboxyl terminals.

Antigenic epitope-bearing peptides and polypeptides of the invention aretherefore useful to raise antibodies, including monoclonal antibodies,that bind specifically to a polypeptide of the invention. See, forinstance, Wilson et al., Cell 37:767-778 (1984) at 777. Antigenicepitope-bearing peptides and polypeptides of the invention preferablycontain a sequence of at least seven, more preferably at least nine andmost preferably between at least about 15 to about 30 amino acidscontained within the amino acid sequence of a polypeptide of theinvention.

Non-limiting examples of antigenic polypeptides or peptides that can beused to generate TR9 receptor-specific antibodies include: a polypeptidecomprising amino acid residues from about 4 to about 81 in SEQ ID NO:2,about 116 to about 271 in SEQ ID NO:2, about 283 to about 308 in SEQ IDNO:2, about 336 to about 372 in SEQ ID NO:2, about 393 to about 434 inSEQ ID NO:2, about 445 to about 559 in SEQ ID NO:2, and about 571 toabout 588 in SEQ ID NO:2. As indicated above, the inventors havedetermined that the above polypeptide fragments are antigenic regions ofthe TR9 receptor protein.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means. R. A. Houghten, “General Method forthe Rapid Solid-Phase Synthesis of Large Numbers of Peptides:Specificity of Antigen-Antibody Interaction at the Level of IndividualAmino Acids,” Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985). This“Simultaneous Multiple Peptide Synthesis (SMPS)” process is furtherdescribed in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).

As one of skill in the art will appreciate, TR9 receptor polypeptides ofthe present invention and the epitope-bearing fragments thereofdescribed above can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. This has been shown, e.g., for chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84-86(1988)). Fusion proteins that have a disulfide-linked dimeric structuredue to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric TR9 protein or proteinfragment alone (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)).

Polypeptide Assays

The present invention also relates to diagnostic assays such asquantitative and diagnostic assays for detecting levels of TR9 receptorprotein, or the soluble form thereof, in cells and tissues, includingdetermination of normal and abnormal levels. Thus, for instance, adiagnostic assay in accordance with the invention for detectingover-expression of TR9, or soluble form thereof, compared to normalcontrol tissue samples may be used to detect the presence of tumors, forexample. Assay techniques that can be used to determine levels of aprotein, such as a TR9 protein of the present invention, or a solubleform thereof, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

Assaying TR9 protein levels in a biological sample can occur using anyart-known method. By “biological sample” is intended any biologicalsample obtained from an individual, cell line, tissue culture, or othersource which contains TR9 receptor protein or mRNA. Preferred forassaying TR9 protein levels in a biological sample are antibody-basedtechniques. For example, TR9 protein expression in tissues can bestudied with classical immunohistological methods. (Jalkanen et al., J.Cell. Biol. 101:976-985 (1985); Jalkanen et al., J. Cell. Biol.105:3087-3096 (1987)). Other antibody-based methods useful for detectingTR9 receptor gene expression include immunoassays, such as the enzymelinked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).

Suitable labels are known in the art and include enzyme labels, such as,glucose oxidase, and radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon(¹⁴C), sulphur (³⁵S), tritium (³H), indium (¹¹²In), and technetium(^(99m)Tc), and fluorescent labels, such as fluorescein and rhodamine,and biotin.

Therapeutics

The tumor necrosis factor (TNF) family ligands are known to be among themost pleiotropic cytokines, inducing a large number of cellularresponses, including cytotoxicity, anti-viral activity, immunoregulatoryactivities, and the transcriptional regulation of several genes (D. V.Goeddel et al., “Tumor Necrosis Factors: Gene Structure and BiologicalActivities,” Symp. Quant. Biol. 51:597-609 (1986), Cold Spring Harbor;B. Beutler and A. Cerami, Annu. Rev. Biochem. 57:505-518 (1988); L. J.Old, Sci. Am. 258:59-75 (1988); W. Fiers, FEBS Lett. 285:199-224(1991)). The TNF-family ligands induce such various cellular responsesby binding to TNF-family receptors, including the TR9 of the presentinvention. Cells which express the TR9 polypeptide and are believed tohave a potent cellular response to TR9 ligands include fetal liver, PBL,lung, kidney, small intestine, colon, keratinocytes, endothelial cells,and monocyte activated tissue. By “a cellular response to a TNF-familyligand” is intended any genotypic, phenotypic, and/or morphologic changeto a cell, cell line, tissue, tissue culture or patient that is inducedby a TNF-family ligand. As indicated, such cellular responses includenot only normal physiological responses to TNF-family ligands, but alsodiseases associated with increased apoptosis or the inhibition ofapoptosis. Apoptosis-programmed cell death-is a physiological mechanisminvolved in the deletion of peripheral T lymphocytes of the immunesystem, and its dysregulation can lead to a number of differentpathogenic processes (J. C. Ameisen, AIDS 8:1197-1213 (1994); P. H.Krammer et al., Curr. Opin. Immunol. 6:279-289 (1994)).

Diseases associated with increased cell survival, or the inhibition ofapoptosis, include cancers (such as follicular lymphomas, carcinomaswith p53 mutations, and hormone-dependent tumors, such as breast cancer,prostrate cancer, Kaposi's sarcoma and ovarian cancer); autoimmunedisorders (such as systemic lupus erythematosus and immune-relatedglomerulonephritis rheumatoid arthritis); viral infections (such asherpes viruses, pox viruses and adenoviruses); inflammation; graft vs.host disease; acute graft rejection and chronic graft rejection.Diseases associated with increased apoptosis include AIDS;neurodegenerative disorders (such as Alzheimer's disease, Parkinson'sdisease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellardegeneration); myelodysplastic syndromes (such as aplastic anemia),ischemic injury (such as that caused by myocardial infarction, strokeand reperfusion injury), toxin-induced liver disease (such as thatcaused by alcohol), septic shock, cachexia and anorexia.

Thus, in one aspect, the present invention is directed to a method forenhancing apoptosis induced by a TNF-family ligand, which involvesadministering to a cell which expresses the TR9 polypeptide, aneffective amount of TR9 ligand, analog or an agonist capable ofincreasing TR9 mediated signaling. Preferably, TR9 mediated signaling isincreased to treat a disease wherein decreased apoptosis or decreasedcytokine and adhesion molecule expression is exhibited. Agonistsinclude, but are not limited to, soluble forms of TR9 and antibodies(preferably monoclonal) directed against the TR9 polypeptide.

In a further aspect, the present invention is directed to a method forinhibiting apoptosis induced by a TNF-family ligand, which involvesadministering to a cell which expresses the TR9 polypeptide an effectiveamount of an antagonist capable of decreasing TR9 mediated signaling.Preferably, TR9 mediated signaling is decreased to treat a diseasewherein increased apoptosis, NFkB expression and/or JNK expression isexhibited. Antagonists include, but are not limited to, soluble forms ofTR9 polypeptide and antibodies (preferably monoclonal) directed againstthe TR9 polypeptide.

By “agonist” is intended naturally occurring and synthetic compoundscapable of enhancing or potentiating apoptosis. By “antagonist” isintended naturally occurring and synthetic compounds capable ofinhibiting apoptosis. Whether any candidate “agonist” or “antagonist” ofthe present invention can enhance or inhibit apoptosis can be determinedusing art-known TNF-family ligand/receptor cellular response assays,including those described in more detail below.

One such screening procedure involves the use of melanophores which aretransfected to express the receptor of the present invention. Such ascreening technique is described in PCT WO 92/01810, published Feb. 6,1992. Such an assay may be employed, for example, for screening for acompound which inhibits (or enhances) activation of the receptorpolypeptide of the present invention by contacting the melanophore cellswhich encode the receptor with both a TNF-family ligand and thecandidate antagonist (or agonist). Inhibition or enhancement of thesignal generated by the ligand indicates that the compound is anantagonist or agonist of the ligand/receptor signaling pathway.

Other screening techniques include the use of cells which express thereceptor (for example, transfected CHO cells) in a system which measuresextracellular pH changes caused by receptor activation, for example, asdescribed in Science 246:181-296 (1989). For example, compounds may becontacted with a cell which expresses the receptor polypeptide of thepresent invention and a second messenger response, e.g., signaltransduction or pH changes, may be measured to determine whether thepotential compound activates or inhibits the receptor.

Another such screening technique involves introducing RNA encoding thereceptor into Xenopus oocytes to transiently express the receptor. Thereceptor oocytes may then be contacted with the receptor ligand and acompound to be screened, followed by detection of inhibition oractivation of a calcium signal in the case of screening for compoundswhich are thought to inhibit activation of the receptor.

Another screening technique well known in the art involves expressing incells a construct wherein the receptor is linked to a phospholipase C orD. Exemplary cells include endothelial cells, smooth muscle cells,embryonic kidney cells, etc. The screening may be accomplished ashereinabove described by detecting activation of the receptor orinhibition of activation of the receptor from the phospholipase signal.

Another method involves screening for compounds which inhibit activationof the receptor polypeptide of the present invention antagonists bydetermining inhibition of binding of labeled ligand to cells which havethe receptor on the surface thereof. Such a method involves transfectinga eukaryotic cell with DNA encoding the receptor such that the cellexpresses the receptor on its surface and contacting the cell with acompound in the presence of a labeled form of a known ligand. The ligandcan be labeled, e.g., by radioactivity. The amount of labeled ligandbound to the receptors is measured, e.g., by measuring radioactivity ofthe receptors. If the compound binds to the receptor as determined by areduction of labeled ligand which binds to the receptors, the binding oflabeled ligand to the receptor is inhibited.

Soluble forms of the polypeptides of the present invention may beutilized in the ligand binding assay described above. These forms of theTR9 receptors are contacted with ligands in the extracellular mediumafter they are secreted. A determination is then made as to whether thesecreted protein will bind to TR9 receptor ligands.

Further screening assays for agonists and antagonists of the presentinvention are described in Tartaglia et al., J. Biol. Chem.267:4304-4307(1992).

Thus, in a further aspect, a screening method is provided fordetermining whether a candidate agonist or antagonist is capable ofenhancing or inhibiting a cellular response to a TNF-family ligand. Themethod involves contacting cells which express the TR9 polypeptide witha candidate compound and a TNF-family ligand, assaying a cellularresponse, and comparing the cellular response to a standard cellularresponse, the standard being assayed when contact is made with theligand in absence of the candidate compound, whereby an increasedcellular response over the standard indicates that the candidatecompound is an agonist of the ligand/receptor signaling pathway and adecreased cellular response compared to the standard indicates that thecandidate compound is an antagonist of the ligand/receptor signalingpathway. By “assaying a cellular response” is intended qualitatively orquantitatively measuring a cellular response to a candidate compoundand/or a TNF-family ligand (e.g., determining or estimating an increaseor decrease in T cell proliferation or tritiated thymidine labeling). Bythe invention, a cell expressing the TR9 polypeptide can be contactedwith either an endogenous or exogenously administered TNF-family ligand.

Agonist according to the present invention include naturally occurringand synthetic compounds such as, for example, TNF family ligand peptidefragments, transforming growth factor, neurotransmitters (such asglutamate, dopamine, N-methyl-D-aspartate), tumor suppressors (p53),cytolytic T cells and antimetabolites. Preferred agonists includechemotherapeutic drugs such as, for example, cisplatin, doxorubicin,bleomycin, cytosine arabinoside, nitrogen mustard, methotrexate andvincristine. Others include ethanol and -amyloid peptide. (Science267:1457-1458 (1995)). Further preferred agonists include polyclonal andmonoclonal antibodies raised against the TR9 polypeptides of theinvention, or a fragment thereof. Such agonist antibodies raised againsta TNF-family receptor are disclosed in Tartaglia et al., Proc. Natl.Acad. Sci. USA 88:9292-9296 (1991); and Tartaglia et al., J. Biol. Chem.267:4304-4307(1992). See, also, PCT Application WO 94/09137.

Antagonists according to the present invention include naturallyoccurring and synthetic compounds such as, for example, the CD40 ligand,neutral amino acids, zinc, estrogen, androgens, viral genes (such asAdenovirus ElB, Baculovirus p35 and IAP, Cowpox virus crmA, Epstein-Barrvirus BHRF1, LMP-1, African swine fever virus LMW5-HL, and Herpesvirusyl 34.5), calpain inhibitors, cysteine protease inhibitors, and tumorpromoters (such as PMA, Phenobarbital, and □-Hexachlorocyclohexane).

In specific embodiments, antagonists according to the present inventionare nucleic acids corresponding to the sequences contained in FIGS.1A-D, or the complementary strand thereof, and/or to nucleotidesequences contained in the deposited clone. In one embodiment, antisensesequence is generated internally by the organism, in another embodiment,the antisense sequence is separately administered (see, for example,O'Connor, J., Neurochem. 56:560 (1991). Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988). Antisense technology can be used to control gene expressionthrough antisense DNA or RNA, or through triple-helix formation.Antisense techniques are discussed for example, in Okano, J., Neurochem.56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance, Lee et al., Nucleic Acids Research 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1300 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA.

For example, the 5′ coding portion of a polynucleotide that encodes themature polypeptide of the present invention may be used to design anantisense RNA polynucleotide of from about 10 to 40 base pairs inlength. A DNA polynucleotide is designed to be complementary to a regionof the gene involved in transcription thereby preventing transcriptionand the production of the receptor. The antisense RNA polypeptidehybridizes to the mRNA in vivo and blocks translation of the mRNAmolecule into receptor polypeptide. The polynucleotides described hereincan also be delivered to cells such that the antisense RNA or DNA may beexpressed in vivo to inhibit production of the receptor.

In one embodiment, the TR9 antisense nucleic acid of the invention isproduced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the TR9 antisense nucleic acid. Such avector can remain episomal or become chromosomally integrated, as longas it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others know inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding TR9, or fragments thereof, can be byany promoter known in the art to act in vertebrate, preferably humancells. Such promoters can be inducible or constitutive. Such promotersinclude, but are not limited to, the SV40 early promoter region(Bernoist and Chambon, Nature 29:304-310 (1981), the promoter containedin the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al.,Cell 22:787-797 (1980), the herpes thymidine promoter (Wagner et al.,Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445 (1981), the regulatorysequences of the metallothionein gene (Brinster et al., Nature 296:39-42(1982)), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a TR9 gene.However, absolute complementarity, although preferred, is not required.A sequence “complementary to at least a portion of an RNA,” referred toherein, means a sequence having sufficient complementarity to be able tohybridize with the RNA, forming a stable duplex; in the case of doublestranded TR9 antisense nucleic acids, a single strand of the duplex DNAmay thus be tested, or triplex formation may be assayed. The ability tohybridize will depend on both the degree of complementarity and thelength of the antisense nucleic acid Generally, the larger thehybridizing nucleic acid, the more base mismatches with a TR9 RNA it maycontain and still form a stable duplex (or triplex as the case may be).One skilled in the art can ascertain a tolerable degree of mismatch byuse of standard procedures to determine the melting point of thehybridized complex.

Potential antagonists according to the invention also include catalyticRNA, or a ribozyme (See, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; Sarver et al, Science 247:1222-1225(1990). While ribozymes that cleave mRNA at site specific recognitionsequences can be used to destroy TR9 mRNAs, the use of hammerheadribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locationsdictated by flanking regions that form complementary base pairs with thetarget mRNA. The sole requirement is that the target mRNA have thefollowing sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Haseloff and Gerlach, Nature 334:585-591 (1988).There are numerous potential hammerhead ribozyme cleavage sites withinthe nucleotide sequence of TR9 (FIGS. 1A-D). Preferably, the ribozyme isengineered so that the cleavage recognition site is located near the 5′end of the TR9 mRNA; i.e., to increase efficiency and minimize theintracellular accumulation of non-functional mRNA transcripts. DNAconstructs encoding the ribozyme may be introduced into the cell in thesame manner as described above for the introduction of antisenseencoding DNA. Since ribozymes, unlike antisense molecules are catalytic,a lower intracellular concentration is required for efficiency.

Further antagonists according to the present invention include solubleforms of TR9, (e.g., fragments of the TR9 receptor sequence depicted inFIGS. 1A-D that include the ligand binding domain from the extracellularregion of the full length receptor). Such soluble forms of the receptor,which may be naturally occurring or synthetic, antagonize TR9 mediatedsignaling by competing with the cell surface TR9 for binding toTNF-family ligands. Thus, soluble forms of the receptor that include theligand binding domain are novel cytokines capable of inhibitingapoptosis induced by TNF-family ligands. These are preferably expressedas dimers or trimers, since these have been shown to be superior tomonomeric forms of soluble receptor as antagonists, e.g., IgGFc-TNFreceptor family fusions. Other such cytokines are known in the art andinclude Fas B (a soluble form of the mouse Fas receptor) that actsphysiologically to limit apoptosis induced by Fas ligand (Hughes andCrispe, J. Exp. Med. 182:1395-1401 (1995)).

The experiments set forth in Example 5 and 6, indicate that the TR9receptor, like other homologous proteins, is a death domain-containingmolecule capable of triggering apoptosis, which is important in theregulation of the immune system. In addition, the experiments set forthbelow suggest that TR9-induced apoptosis will be blocked by theinhibitors of ICE-like proteases, CrmA and z-VAD-fmk. Importantly, it isalso expected that apoptosis induced by TR9 will be blocked by dominantnegative versions of FADD (FADD-DN) or FLICE (FLICE-DN/MACHa1C360S),which were previously shown to inhibit death signaling by Fas/APO-1 andTNFR-1. Thus, inhibitors of ICE-like proteases, FADD-DN andFLICE-DN/MACHa1C360S could also be used as antagonists for TR9 activity.

Antagonists of the present invention also include antibodies specificfor TNF-family ligands or the TR9 polypeptides of the invention. Theterm “antibody” (Ab) or “monoclonal antibody” (mAb) as used herein ismeant to include intact molecules as well as fragments thereof (such as,e.g., Fab and F(ab′)₂ fragments) which are capable of binding anantigen. Fab and F(ab′)₂ fragments lack the Fc fragment of intactantibody, clear more rapidly from the circulation, and may have lessnon-specific tissue binding of an intact antibody (Wahl et al., J. Nucl.Med. 24:316-325 (1983)).

Antibodies according to the present invention may be prepared by any ofa variety of standard methods using TR9 immunogens of the presentinvention. As indicated, such TR9 immunogens include the full length TR9polypeptide depicted in FIGS. 1A-D (SEQ ID NO:2) (which may or may notinclude the leader sequence) and TR9 polypeptide fragments comprising,for example, the ligand binding domain, extracellular domain,transmembrane domain, intracellular domain, death domain, incompletedeath domain, or any combination thereof.

Polyclonal and monoclonal antibody agonists or antagonists according tothe present invention can be raised according to the methods disclosedin Tartaglia and Goeddel, J. Biol. Chem. 267(7):4304-4307(1992));Tartaglia et al., Cell 73:213-216 (1993)), and PCT Application WO94/09137 and are preferably specific to (i.e., bind uniquely topolypeptides of the invention having the amino acid sequence of SEQ IDNO:2. The term “antibody” (Ab) or “monoclonal antibody” (mAb) as usedherein is meant to include intact molecules as well as fragments thereof(such as, for example, Fab and F(ab′) fragments) which are capable ofbinding an antigen. Fab, Fab′ and F(ab′) fragments lack the Fc fragmentintact antibody, clear more rapidly from the circulation, and may haveless non-specific tissue binding of an intact antibody (Wahl et al., J.Nucl. Med., 24:316-325 (1983)).

In a preferred method, antibodies according to the present invention aremAbs. Such mAbs can be prepared using hybridoma technology (Kohler andMillstein, Nature 256:495-497 (1975) and U.S. Pat. No. 4,376,110; Harlowet al., Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1988; Monoclonal Antibodies andHybridomas: A New Dimension in Biological Analyses, Plenum Press, NewYork, N.Y., 1980; Campbell, “Monoclonal Antibody Technology,” In:Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13(Burdon et al., eds.), Elsevier, Amsterdam (1984)).

Proteins and other compounds which bind the TR9 domains are alsocandidate agonists and antagonists according to the present invention.Such binding compounds can be “captured” using the yeast two-hybridsystem (Fields and Song, Nature 340:245-246 (1989)). A modified versionof the yeast two-hybrid system has been described by Roger Brent and hiscolleagues (Gyuris, Cell 75:791-803 (1993); Zervos et al., Cell72:223-232 (1993)). Preferably, the yeast two-hybrid system is usedaccording to the present invention to capture compounds which bind tothe ligand binding domain, extracellular, intracellular, transmembrane,and death domain of the TR9. Such compounds are good candidate agonistsand antagonists of the present invention.

Using the two-hybrid assay described above, the intracellular domain ofthe TR9 receptor, or a portion thereof, may be used to identify cellularproteins which interact with the receptor in vivo. Such an assay mayalso be used to identify ligands with potential agonistic orantagonistic activity of TR9 receptor function. This screening assay haspreviously been used to identify protein which interact with thecytoplasmic domain of the murine TNF-RII and led to the identificationof two receptor associated proteins. Rothe et al., Cell 78:681 (1994).Such proteins and amino acid sequences which bind to the cytoplasmicdomain of the TR9 receptors are good candidate agonist and antagonist ofthe present invention.

Other screening techniques include the use of cells which express thepolypeptide of the present invention (for example, transfected CHOcells) in a system which measures extracellular pH changes caused byreceptor activation, for example, as described in Science, 246:181-296(1989). In another example, potential agonists or antagonists may becontacted with a cell which expresses the polypeptide of the presentinvention and a second messenger response, e.g., signal transduction maybe measured to determine whether the potential antagonist or agonist iseffective.

By a “TNF-family ligand” is intended naturally occurring, recombinant,and synthetic ligands that are capable of binding to a member of the TNFreceptor family and inducing the ligand/receptor signaling pathway.Members of the TNF ligand family include, but are not limited to TR9ligands including TRAIL, TNF-α, lymphotoxin-α (LT-α, also known asTNF-β), LT-β (found in complex heterotrimer LT-α2-β), FasL, CD40, CD27,CD30, 4-1BB, OX40, and nerve growth factor (NGF).

Representative therapeutic applications of the present invention arediscussed in-more detail below. The state of immunodeficiency thatdefines AIDS is secondary to a decrease in the number and function ofCD4⁺ T-lymphocytes. Recent reports estimate the daily loss of CD4⁺ Tcells to be between 3.5×10⁷ and 2×10⁹ cells (Wei et al., Nature373:117-122 (1995)). One cause of CD4⁺ T cell depletion in the settingof HIV infection is believed to be HIV-induced apoptosis. Indeed,HIV-induced apoptotic cell death has been demonstrated not only in vitrobut also, more importantly, in infected individuals (Ameisen, J. C.,AIDS 8:1197-1213 (1994); Finkel and Banda, Curr. Opin. Immunol.6:605-615(1995); Muro-Cacho et al., J. Immunol. 154:5555-5566 (1995)).Furthermore, apoptosis and CD4⁺ T-lymphocyte depletion is tightlycorrelated in different animal models of AIDS (Brunner et al., Nature373:441-444 (1995); Gougeon et al., AIDS Res. Hum. Retroviruses9:553-563 (1993)) and, apoptosis is not observed in those animal modelsin which viral replication does not result in AIDS. Id. Further dataindicates that uninfected but primed or activated T lymphocytes fromHIV-infected individuals undergo apoptosis after encountering theTNF-family ligand FasL. Using monocytic cell lines that result in deathfollowing HIV infection, it has been demonstrated that infection of U937cells with HIV results in the de novo expression of FasL and that FasLmediates HIV-induced apoptosis (Badley et al., J. Virol. 70:199-206(1996)). Further, the TNF-family ligand was detectable in uninfectedmacrophages and its expression was upregulated following HIV infectionresulting in selective killing of uninfected CD4 T-lymphocytes. Id.Thus, by the invention, a method for treating HIV⁺ individuals isprovided which involves administering an antagonist of the presentinvention to reduce selective killing of CD4 T-lymphocytes. Modes ofadministration and dosages are discussed in detail below.

In rejection of an allograft, the immune system of the recipient animalhas not previously been primed to respond because the immune system forthe most part is only primed by environmental antigens. Tissues fromother members of the same species have not been presented in the sameway than, for example, viruses and bacteria have been presented. In thecase of allograft rejection, immunosuppressive regimens are designed toprevent the immune system from reaching the effector stage. However, theimmune profile of xenograft rejection may resemble disease recurrencemore than allograft rejection. In the case of disease recurrence, theimmune system has already been activated, as evidenced by destruction ofthe native islet cells. Therefore, in disease recurrence, the immunesystem is already at the effector stage. Agonists of the presentinvention are able to suppress the immune response to both allograftsand xenografts because lymphocytes activated and differentiated intoeffector cells will express the TR9 polypeptide, and thereby aresusceptible to compounds which enhance apoptosis. Thus, the presentinvention further provides a method for creating immune privilegedtissues.

TR9 antagonists of the invention can further be used in the treatment ofinflammatory diseases and stress response related diseases, such asinflammatory bowel disease, rheumatoid arthritis, osteoarthritis,psoriasis, and septicemia.

In addition, due to lymphoblast expression of TR9, soluble TR9 agonistor antagonist antibodies (e.g., mABs) may be used to treat this form ofcancer. Further, soluble TR9 or neutralizing mABs may be used to treatvarious chronic and acute forms of inflammation such as rheumatoidarthritis, osteoarthritis, psoriasis, septicemia, and inflammatory boweldisease.

Modes of Administration

The agonist or antagonists described herein can be administered invitro, ex vivo, or in vivo to cells which express the receptor of thepresent invention. By administration of an “effective amount” of anagonist or antagonist is intended an amount of the compound that issufficient to enhance or inhibit a cellular response to a TNF-familyligand and include polypeptides. In particular, by administration of an“effective amount” of an agonist or antagonists is intended an amounteffective to enhance or inhibit TR9 mediated apoptosis. Of course, whereit is desired for apoptosis to be enhanced, an agonist according to thepresent invention can be co-administered with a TNF-family ligand. Oneof ordinary skill will appreciate that effective amounts of an agonistor antagonist can be determined empirically and may be employed in pureform or in pharmaceutically acceptable salt, ester or prodrug form Theagonist or antagonist may be administered in compositions in combinationwith one or more pharmaceutically acceptable excipients (i.e.,carriers).

It will be understood that, when administered to a human patient, thetotal daily usage of the compounds and compositions of the presentinvention will be decided by the attending physician within the scope ofsound medical judgment. The specific therapeutically effective doselevel for any particular patient will depend upon factors well known inthe medical arts.

As a general proposition, the total pharmaceutically effective amount ofTR9 polypeptide administered parenterally per dose will be in the rangeof about 1 μg/kg/day to 10 mg/kg/day of patient body weight, although,as noted above, this will be subject to therapeutic discretion. Morepreferably, this dose is at least 0.01 mg/kg/day, and most preferablyfor humans between about 0.01 and 1 mg/kg/day for the hormone. If givencontinuously, the TR9 polypeptide is typically administered at a doserate of about 1 μg/kg/hour to about 50 μg/kg/hour, either by 1-4injections per day or by continuous subcutaneous infusions, for example,using a mini-pump. An intravenous bag solution may also be employed.

Dosaging may also be arranged in a patient specific manner to provide apredetermined concentration of an agonist or antagonist in the blood, asdetermined by the RIA technique. Thus patient dosaging may be adjustedto achieve regular on-going trough blood levels, as measured by RIA, onthe order of from 50 to 1000 ng/ml, preferably 150 to 500 ng/ml.

Pharmaceutical compositions are provided comprising an agonist(including TR9 receptor polynucleotides, polypeptides or antibodies ofthe invention) or agonist (e.g., TR9 polynucleotides, polypeptides ofthe invention or antibodies thereto) of TR9 and a pharmaceuticallyacceptable carrier or excipient, which may be administered orally,rectally, parenterally, intracistemally, intravaginally,intraperitoneally, topically (as by powders, ointments, drops ortransdermal patch), bucally, or as an oral or nasal spray, In oneembodiment “pharmaceutically acceptable carrier” means a non-toxicsolid, semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. In a specific embodiment,“pharmaceutically acceptable” means approved by a regulatory agency ofthe federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly humans. Nonlimiting examples of suitable pharmaceuticalcarriers according to this embodiment are provided in “Remington'sPharmaceutical Sciences” by E. W. Martin, and include sterile liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin, such as peanut oil, soybean oil, mineral oil,sesame oil and the like. Water is a preferred carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can be employed asliquid carriers, particularly for injectable solutions.

The term “parenteral” as used herein refers to modes of administrationwhich include intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intraarticular injection and infusion.

Pharmaceutical compositions of the present invention for parenteralinjection can comprise pharmaceutically acceptable sterile aqueous ornonaqueous solutions, dispersions, suspensions or emulsions as well assterile powders for reconstitution into sterile injectable solutions ordispersions just prior to use.

In addition to soluble TR9 polypeptides, TR9 polypeptides containing thetransmembrane region can also be used when appropriately solubilized byincluding detergents, such as CHAPS or NP-40, with buffer.

Chromosome Assays

The nucleic acid molecules of the present invention are also valuablefor chromosome identification. The sequence is specifically targeted toand can hybridize with a particular location on an individual humanchromosome. The mapping of DNAs to chromosomes according to the presentinvention is an important first step in correlating those sequences withgenes associated with disease.

In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a TR9 receptor gene. This canbe accomplished using a variety of well known techniques and libraries,which generally are available commercially. The genomic DNA then is usedfor in situ chromosome mapping using well known techniques for thispurpose.

In addition, in some cases, sequences can be mapped to chromosomes bypreparing PCR primers (preferably 15-25 bp) from the cDNA. Computeranalysis of the 3′ untranslated region of the gene is used to rapidlyselect primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes.

Fluorescence in situ hybridization (“FISH”) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with probesfrom the cDNA as short as 50 or 60 bp. For a review of this technique,see Verma et al., Human Chromosomes: A Manual Of Basic Techniques,Pergamon Press, N.Y. (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance In Man, available on-line through Johns HopkinsUniversity, Welch Medical Library. The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Expression and Purification of the TR9 Receptor in E.coli

The bacterial expression vector pQE60 is used for bacterial expressionin this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif.,91311). pQE60 encodes ampicillin antibiotic resistance (“Amp^(r)”) andcontains a bacterial origin of replication (“ori”), an IPTG induciblepromoter, a ribosome binding site (“RBS”), six codons encoding histidineresidues that allow affinity purification usingnickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin sold by QIAGEN,Inc., supra, and suitable single restriction enzyme cleavage sites.These elements are arranged such that a DNA fragment encoding apolypeptide may be inserted in such as way as to produce thatpolypeptide with the six His residues (i.e., a “6× His tag”) covalentlylinked to the carboxyl terminus of that polypeptide. However, in thisexample, the polypeptide coding sequence is inserted such thattranslation of the six His codons is prevented and, therefore, thepolypeptide is produced with no 6× His tag.

The DNA sequence encoding the desired portion of the TR9 receptorprotein lacking the hydrophobic leader sequence is amplified from thedeposited cDNA clone using PCR oligonucleotide primers which anneal tothe amino terminal sequences of the desired portion of the TR9 receptorprotein and to sequences in the deposited construct 3′ to the cDNAcoding sequence. Additional nucleotides containing restriction sites tofacilitate cloning in the pQE60 vector are added to the 5′ and 3′sequences, respectively.

For cloning the mature protein, the 5′ primer has the sequence:

5′ CGCCCATGGCTCAGCCAGAACAGAAG 3′ (SEQ ID NO:11) containing theunderlined NcoI restriction site followed by 17 nucleotidescomplementary to the amino terminal coding sequence of the mature TR9receptor sequence in FIGS. 1A-D. One of ordinary skill in the art wouldappreciate, of course, that the point in the protein coding sequencewhere the 5′ primer begins may be varied to amplify a desired portion ofthe complete protein shorter or longer than the mature form. The 3′primer has the sequence:

5′ CGCAAGCTTTTAGGGCAAATGCTCATTG3′ (SEQ ID NO:12) containing theunderlined HindIII restriction site followed by 19 nucleotidescomplementary to the 3′ end of the non-coding sequence in the TR9receptor DNA sequence in FIGS. 1A-D.

The amplified TR9 receptor DNA fragments and the vector pQE60 aredigested with NcoI and HindIII, and the digested DNAs are then ligatedtogether. Insertion of the TR9 receptor DNA into the restricted pQE60vector places the TR9 receptor protein coding region including itsassociated stop codon downstream from the IPTG-inducible promoter andin-frame with an initiating AUG. The associated stop codon preventstranslation of the six histidine codons downstream of the insertionpoint.

The ligation mixture is transformed into competent E. coli cells usingstandard procedures such as those described in Sambrook et al.,Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses the lac repressor and confers kanamycin resistance(“Kan^(r)”), is used in carrying out the illustrative example describedherein. This strain, which is only one of many that are suitable forexpressing TR9 receptor protein, is available commercially from QIAGEN,Inc., supra. Transformants are identified by their ability to grow on LBplates in the presence of ampicillin and kanamycin. Plasmid DNA isisolated from resistant colonies and the identity of the cloned DNAconfirmed by restriction analysis, PCR and DNA sequencing.

Clones containing the desired constructs are grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml). The O/N culture is used to inoculate a largeculture, at a dilution of approximately 1:25 to 1:250. The cells aregrown to an optical density at 600 nm (“OD600”) of between 0.4 and 0.6.Isopropyl-b-D-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from the lac repressorsensitive promoter, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation.

The cells are then stirred for 34 hours at 4° C. in 6M guanidine-HCl,pH8. The cell debris is removed by centrifugation, and the supernatantcontaining the TR9 receptor is dialyzed against 50 mM Na-acetate bufferpH6, supplemented with 200 mM NaCl. Alternatively, the protein can besuccessfully refolded by dialyzing it against 500 mM NaCl, 20% glycerol,25 mM Tris/HCl pH7.4, containing protease inhibitors. After renaturationthe protein can be purified by ion exchange, hydrophobic interaction andsize exclusion chromatography. Alternatively, an affinity chromatographystep such as an antibody column can be used to obtain pure TR9 receptorprotein. The purified protein is stored at 4° C. or frozen at −80° C.

Example 2 Cloning and Expression of the TR9 Receptor Protein in aBaculovirus Expression System

In this illustrative example, the plasmid shuttle vector pA2 is used toinsert the cloned DNA encoding the complete protein, including itsnaturally associated secretary signal (leader) sequence, into abaculovirus to express the mature TR9 receptor protein, using standardmethods as described in Summers et al., A Manual of Methods forBaculovirus Vectors and Insect Cell Culture Procedures, TexasAgricultural Experimental Station Bulletin No. 1555 (1987). Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed byconvenient restriction sites such as BamHI and Asp718. Thepolyadenylation site of the simian virus 40 (“SV40”) is used forefficient polyadenylation. For easy selection of recombinant virus, theplasmid contains the beta-galactosidase gene from E. coli under controlof a weak Drosophila promoter in the same orientation, followed by thepolyadenylation signal of the polyhedrin gene. The inserted genes areflanked on both sides by viral sequences for cell-mediated homologousrecombination with wild-type viral DNA to generate viable virus thatexpress the cloned polynucleotide.

Many other baculovirus vectors could be used in place of the vectorabove, such as pAc373, pVL941 and pAcIM1, as one skilled in the artwould readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31-39 (1989).

The cDNA sequence encoding the fall length TR9 protein in the depositedclone, including the AUG initiation codon and the naturally associatedleader sequence shown in FIGS. 1A-D (SEQ ID NO:2), is amplified usingPCR oligonucleotide primers corresponding to the 5′ and 3′ sequences ofthe gene. The 5′ primer has the sequence: 5′CGCCCCGGGGCCATCATGGGGACCTCTCCGAGC 3′ (SEQ ID NO:13) containing theunderlined SmaI restriction enzyme site, an efficient signal forinitiation of translation in eukaryotic cells, as described by Kozak,M., J. Mol. Biol. 196:947-950 (1987), followed by a number of bases ofthe sequence of the complete TR9 receptor protein shown in FIGS. 1A-D,beginning with the AUG initiation codon.

The 3′ primer (for cloning the soluble form) has the sequence: 5′CGCGGTACCTTAGGGCAAAT GCTCATTG 3′ (SEQ ID NO:14) containing theunderlined Asp718 restriction site followed by nucleotides complementaryto the 3′ noncoding sequence in FIGS. 1A-D.

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with SmaI and Asp718 and again ispurified on a 1% agarose gel. This fragment is designated herein “F1”.

The plasmid is digested with the restriction enzymes SmaI and Asp718 andoptionally, can be dephosphorylated using calf intestinal phosphatase,using routine procedures known in the art. The DNA is then isolated froma 1% agarose gel using a commercially available kit (“Geneclean” BIO 101Inc., La Jolla, Calif.). This vector DNA is designated herein “V1”.

Fragment F1 and the dephosphorylated plasmid V1 are ligated togetherwith T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts suchas XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells aretransformed with the ligation mixture and spread on culture plates.Bacteria are identified that contain the plasmid with the human TR9receptor gene using the PCR method, in which one of the primers that isused to amplify the gene and the second primer is from well within thevector so that only those bacterial colonies containing the TR9 receptorgene fragment will show amplification of the DNA. The sequence of thecloned fragment is confirmed by DNA sequencing. This plasmid isdesignated herein pBacTR9.

Five μg of the plasmid pBacTR9 are co-transfected with 1.0 μg of acommercially available linearized baculovirus DNA (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner et al., Proc. Natl. Acad. Sci. USA84:7413-7417 (1987). One μg of BaculoGold™ virus DNA and 5 μg of theplasmid pBacTR9 are mixed in a sterile well of a microtiter platecontaining 50 μl of serum-free Grace's medium (Life Technologies Inc.,Rockville, Md.). Afterwards, 10 μl Lipofectin plus 90 μl Grace's mediumare added, mixed and incubated for 15 minutes at room temperature. Thenthe transfection mixture is added drop-wise to Sf9 insect cells (ATCCCRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace'smedium without serum. The plate is rocked back and forth to mix thenewly added solution. The plate is then incubated for 5 hours at 27° C.After 5 hours the transfection solution is removed from the plate and 1ml of Grace's insect medium supplemented with 10% fetal calf serum isadded. The plate is put back into an incubator and cultivation iscontinued at 27° C. for four days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, supra. An agarose gel with“Blue Gal” (Life Technologies Inc., Rockville, Md.) is used to alloweasy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Rockville, Md., page 9-10). After appropriate incubation, blue stainedplaques are picked with the tip of a micropipettor (e.g., Eppendorf).The agar containing the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 μl of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. Therecombinant virus is called V-TR9.

To verify the expression of the V-TR9 gene, Sf9 cells are grown inGrace's medium supplemented with 10% heat inactivated FBS. The cells areinfected with the recombinant baculovirus V-TR9 at a multiplicity ofinfection (“MOI”) of about 2. Six hours later the medium is removed andis replaced with SF900 II medium minus methionine and cysteine(available from Life Technologies Inc., Rockville, Md.). If radiolabeledproteins are desired, 42 hours later, 5 μCi of ³⁵S-methionine and 5 μCi³⁵S-cysteine (available from Amersham) are added. The cells are furtherincubated for 16 hours and then they are harvested by centrifugation.The proteins in the supernatant as well as the intracellular proteinsare analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).Microsequencing of the amino acid sequence of the amino terminus ofpurified protein may be used to determine the amino terminal sequence ofthe mature protein and thus the cleavage point and length of thesecretory signal peptide.

Example 3 Cloning and Expression of TR9 in Mammalian Cells

A typical mammalian expression vector contains the promoter element,which mediates the initiation of transcription of mRNA, the proteincoding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as PSVL and PMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude, human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. The co-transfection witha selectable marker such as dhfr, gpt, neomycin, or hygromycin allowsthe identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded protein. The dihydrofolate reductase (DHFR) marker is usefulto develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS) (Murphy et al., Biochem J.227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) and NSO cells are often used for theproduction of proteins.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen et al., Molec. Cell. Biol. 5:438-447(1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell41:521-530 (1985)). Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning ofthe gene of interest. The vectors contain in addition the 3′ intron, thepolyadenylation and termination signal of the rat preproinsulin gene.

Example 3(a) Cloning and Expression in COS Cells

The expression plasmid, pTR9-HA, is made by cloning a cDNA encoding TR9into the expression vector pcDNAI/Amp or pcDNAIII (which can be obtainedfrom Invitrogen, Inc.).

The expression vector pcDNAI/Amp contains: (1) an E. coli origin ofreplication effective for propagation in E. coli and other prokaryoticcells; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron; (5) several codons encoding a hemagglutinin fragment(i.e., an “HA” tag to facilitate purification) followed by a terminationcodon and polyadenylation signal arranged so that a cDNA can beconveniently placed under expression control of the CMV promoter andoperably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker. The HA tag corresponds toan epitope derived from the influenza hemagglutinin protein described byWilson et al., Cell 37:767-778 (1984). The fusion of the HA tag to thetarget protein allows easy detection and recovery of the recombinantprotein with an antibody that recognizes the HA epitope. pcDNAIIIcontains, in addition, the selectable neomycin marker.

A DNA fragment encoding the TR9 is cloned into the polylinker region ofthe vector so that recombinant protein expression is directed by the CMVpromoter. The plasmid construction strategy is as follows. The TR9 cDNAof the deposited clone is amplified using primers that containconvenient restriction sites, much as described above for constructionof vectors for expression of TR9 in E. coli. Suitable primers includethe following, which are used in this example.

The 5′ primer, containing the underlined SmaI site, a Kozak sequence, anAUG start codon and codons of the 5′ coding region of the complete TR9receptor has the following sequence: 5′ CGCCCCGGGGCCATCATGGGGACCTCTCCGAGC 3′ (SEQ ID NO:13).

The 3′ primer, containing the underlined XbaI site, a stop codon, andnucleotides of the 3′ coding sequence, has the following sequence (atthe 3′ end): 5′CGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTAGGGCAAATGCTCATTG3′ (SEQ ID NO:15).

The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digestedwith SmaI and XbaI and then ligated. The ligation mixture is transformedinto E. coli strain SURE (available from Stratagene Cloning Systems,11099 North Torrey Pines Road, La Jolla, Calif. 92037), and thetransformed culture is plated on ampicillin media plates which then areincubated to allow growth of ampicillin resistant colonies. Plasmid DNAis isolated from resistant colonies and examined by restriction analysisor other means for the presence of the TR9-encoding fragment.

For expression of recombinant TR9, COS cells are transfected with anexpression vector, as described above, using DEAE-DEXTRAN, as described,for instance, in Sambrook et al., Molecular Cloning: a LaboratoryManual, Cold Spring Laboratory Press, Cold Spring Harbor, N.Y. (1989).Cells are incubated under conditions for expression of TR9 by thevector.

Expression of the TR9-HA fusion protein is detected by radiolabeling andimmunoprecipitation, using methods described in, for example Harlow etal., Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1988). To this end, two daysafter transfection, the cells are labeled by incubation in mediacontaining ³⁵S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and lysed with detergent-containingRIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM TRIS, pH7.5, as described by Wilson et al. cited above. Proteins areprecipitated from the cell lysate and from the culture media using anHA-specific monoclonal antibody. The precipitated proteins then areanalyzed by SDS-PAGE and autoradiography. An expression product of theexpected size is seen in the cell lysate, which is not seen in negativecontrols.

Example 3(b) Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of TR9 protein. Plasmid pC4 isa derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146). Theplasmid contains the mouse DHFR gene under control of the SV40 earlypromoter. Chinese hamster ovary- or other cells lacking dihydrofolateactivity that are transfected with these plasmids can be selected bygrowing the cells in a selective medium (alpha minus MEM, LifeTechnologies, Rockville, Md.) supplemented with the chemotherapeuticagent methotrexate. The amplification of the DHFR genes in cellsresistant to methotrexate (MTX) has been well documented (see, e.g., Altet al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin et. al., Biochem. etBiophys. Acta, 1097:107-143 (1990); and Page et. al., Biotechnology9:64-68 (1991)). Cells grown in increasing concentrations of MTX developresistance to the drug by overproducing the target enzyme, DHFR, as aresult of amplification of the DHFR gene. If a second gene is linked tothe DHFR gene, it is usually co-amplified and over-expressed. It isknown in the art that this approach may be used to develop cell linescarrying more than 1,000 copies of the amplified gene(s). Subsequently,when the methotrexate is withdrawn, cell lines are obtained whichcontain the amplified gene integrated into one or more chromosome(s) ofthe host cell.

Plasmid pC4 contains for expressing the gene of interest the strongpromoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus(Cullen et al., Molec. Cell. Biol. 5:438-447 (1985)) plus a fragmentisolated from the enhancer of the immediate early gene of humancytomegalovirus (CMV) (Boshart et al., Cell 41:521-530 (1985)).Downstream of the promoter are BamHI, XbaI, and Asp718 restrictionenzyme cleavage sites that allow integration of the genes. Behind thesecloning sites the plasmid contains the 3′ intron and polyadenylationsite of the rat preproinsulin gene. Other high efficiency promoters canalso be used for the expression, e.g., the human β-actin promoter, theSV40 early or late promoters or the long terminal repeats from otherretroviruses, e.g., HIV and HTLVI. Clontech's Tet-Off and Tet-On geneexpression systems and similar systems can be used to express the TR9 ina regulated way in mammalian cells (Gossen et. al., Proc. Natl. Acad.Sci. USA 89:5547-5551 (1992). For the polyadenylation of the mRNA othersignals, e.g., from the human growth hormone or globin genes can be usedas well. Stable cell lines carrying a gene of interest integrated intothe chromosomes can also be selected upon co-transfection with aselectable marker such as gpt, G418 or hygromycin. It is advantageous touse more than one selectable marker in the beginning, e.g., G418 plusmethotrexate.

The plasmid pC4 is digested with the restriction enzymes SmaI and Asp718and then dephosphorylated using calf intestinal phosphatase byprocedures known in the art. The vector is then isolated from a 1%agarose gel.

The DNA sequence encoding the complete TR9 protein including its leadersequence is amplified using PCR oligonucleotide primers corresponding tothe 5′ and 3′ sequences of the gene.

The 5′ primer has the sequence: 5′ CGCCCCGGGGCCATCATGGGGACCTCTCCGAGC 3′(SEQ ID NO:13) restriction enzyme site, an efficient signal forinitiation of translation in eukaryotic cells, as described by Kozak,M., J. Mol. Biol. 196:947-950 (1987), followed by a number of bases ofthe coding sequence of the TR9 receptor protein shown in FIGS. 1A-D (SEQID NO:1).

The 3′ primer (for cloning the soluble form) has the sequence: 5′CGCGGTACCTTAGGGCAAAT GCTCATTG 3′ (SEQ ID NO:14) containing theunderlined Asp718 restriction site followed by nucleotides complementaryto the non-translated region of the TR9 receptor gene shown in FIGS.1A-D (SEQ ID NO:1).

The amplified fragment is digested with the endonucleases SmaI and thenpurified again on a 1% agarose gel. The isolated fragment and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC4 using,for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene are used fortransfection. Five μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSV2-neo using lipofectin (Felgner et al.,supra). The plasmid pSV2neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 μM, 20 μM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reverse phase HPLCanalysis.

Example 4 Tissue Distribution of TR9 mRNA Expression

Northern blot analysis is carried out to examine TR9 gene expression inhuman tissues, using methods described by, among others, Sambrook etal., supra. A cDNA probe containing the entire nucleotide sequence ofthe TR9 protein (SEQ ID NO: 1) is labeled with ³²P using the rediprime™DNA labeling system (Amersham Life Science), according to manufacturer'sinstructions. After labeling, the probe was purified using a CHROMASPIN-100™ column (Clontech Laboratories, Inc.), according tomanufacturer's protocol number PT1200-1. The purified labeled probe isthen used to examine various human tissues for TR9 mRNA.

Multiple Tissue Northern (MTN) blots containing various human tissues(H) or human immune system tissues (IM) are obtained from Clontech andare examined with the labeled probe using ExpressHyb™ hybridizationsolution (Clontech) according to manufacturer's protocol numberPT1190-1. Following hybridization and washing, the blots are mounted andexposed to film at −70° C. overnight, and films developed according tostandard procedures.

Example 5 TR9 Induced Apoptosis

Overexpression of Fas/APO-1 and TNFR-1 in mammalian cells mimicsreceptor activation (M. Muzio et al., Cell 85:817-827 (1996); M. P.Boldin et al., Cell 85:803-815 (1996)). Thus, this system is utilized tostudy the functional role of TR9. Transient expression of TR9 in MCF7breast carcinoma cells and 293 human embryonic kidney cells isinvestigated for induction of apoptosis.

Experimental Design

Cell death assays are performed essentially as previously described (A.M. Chinnaiyan et al., Cell 81:505-512 (1995); M. P. Boldin et al., J.Biol. Chem. 270: 7795-8 (1995); F. C. Kischkel et al., EMBO 14:5579-5588(1995); A. M. Chinnaiyan et al., J. Biol. Chem. 271:4961-4965 (1996)).Briefly, MCF-7 human breast carcinoma clonal cell lines stablytransfected with either vector alone, a CrmA expression construct (M.Tewari et al., J. Biol. Chem. 270:3255-60 (1995)), or FADD-DN expressionconstruct (A. M. Chinnaiyan et al., J. Biol. Chem. 271:4961-4965 (1996))are transiently transfected with pCMV-TR9-galatosidase in the presenceof a ten-fold excess of pcDNA3 expression constructs encoding theindicated proteins using lipofectamine (GIBCO-BRL). 293 cells arelikewise transfected using the CaPO4 method. The ICE family inhibitorz-VAD-fmk (Enzyme Systems Products, Dublin, Calif.) is added to thecells at a concentration of 10□M, 5 hrs after transfection. 32 hoursfollowing transfection, cells are fixed and stained with X-Gal aspreviously described (A. M. Chinnaiyan et al., Cell 81:505-12 (1995); M.P. Boldin et al., J. Biol. Chem. 270:7795-8 (1995); F. C. Kischkel etal., EMBO 14:5579-5588 (1995)).

Results

The affected cells will display morphological alterations typical ofcells undergoing apoptosis, becoming rounded, condensed, and detachingfrom the dish. Similar to TNFR-1 and Fas/APO-l (M. Muzio et al., Cell85:817-827 (1996); M. P. Boldin et al., Cell 85:803-815 (1996); M.Tewari et al., J. Biol. Chem. 270:3255-60 (1995)), TR9-induced apoptosisis blocked by the inhibitors of ICE-like proteases, CrmA and z-VAD-fmk.

Example 6 Characterization of TR9

Members of the TNF receptor family are crucial modulators ofinflammatory and cellular immune responses, and mediate a variety ofbiological functions, ranging from cell proliferation, differentiationand apoptosis to cell survival (Nagata, S., Cell 88:355-365 (1997);Armitage, R. J., Curr. Opin. Immuno. 6:407-413 (1994); Golstein, P.,Curr. Biol. 7:R750-R753 (1997); Baichwal et al., Curr. Biol. 7:R94-R96.(1997); Smith et al., Cell 76: 959-962 (1994); Anderson et al., Nature390:175-179 (1997); and Cleveland et al., Cell 81:479-482 (1995)). Thisfamily of receptors is characterized by several extracellular,cysteine-rich motifs that compose the ligand binding domain (Armitage,R. J., Curr. Opin. Immuno. 6:407-413 (1994); and Smith et al., Cell 76:959-962 (1994)). Upon ligation by their cognate ligands, these receptorsengage a number of signal transduction pathways, including apoptosis,activation of NF κB and JNK pathways that modulate the expression ofgenes involved in the immune and stress response (Smith et al., Cell 76:959-962 (1994)).

Within the TNF receptor family, six members have emerged as a distinctsubgroup termed death receptors; they contain a cytoplasmic death domainand activation of these receptors leads to engagement of components ofthe cell death pathway (Nagata, S., Cell 88:355-365 (1997); andGolstein, P., Curr. Biol. 7:R750-R753 (1997)). Transmission of the deathsignal is mediated by a series of homophilic protein-proteininteractions involving the death domain and death effector domain thatwas originally defined as being present in the adaptor moleculeFADD/MORT1 and the death protease caspase-8 (Chinnaiyan et al., Semmin.Immunol. 9:66-67 (1997)). For example, when the death receptor CD95/Fasis ligated by cognate ligand or agonist antibody, the adaptor moleculeFADD and the death protease caspase-8 are recruited to the signallingcomplex through interactions involving death and death effector domains,respectively (Chinnaiyan et al., Semmin. Immunol. 9:66-67 (1997); Muzioet al., Cell 85: 817-827 (1996); and Boldin et al., Cell 85:803-815(1996)). On approximation, caspase-8 undergoes an autoactivation,initiating activation of the downstream caspases, cleavage of deathsubstrates and demise of the cell (Muzio et al, J. Biol. Chem.273:2952-2956 (1997); Barinaga, M., Science 280:32-34 (1998); Salvesenet al., Cell 91:443-446 (1997); and Martin et al., Cell 82: 349-352(1995)). In contrast to CD-95 that directly engages the FADD-caspase-8pathway (Muzio et al., Cell 85: 817-827 (1996); Boldin et al., Cell85:803-815 (1996); Chinnaiyan et al., Cell 81:505-512 (1995); and Boldinet al., J. Biol. Chem. 270:7795-7789 (1995)), both TNFR1 and DR3 utilizea primary adaptor molecule termed TRADD, around which assembles theFADD-caspase-8 pathway, an NF κB activating pathway involving the deathdomain-containing Ser/Thr kinase RIP and a JNK activating pathway thatis mediated by the adaptor molecule TRAF2 (Hsu et al., Cell 81:495-504(1995); Hsu et al., Immunity 4:387-396 (1996); Chinnaiyan et al.,Science 274:990-992 (1996); Kitson et al., Nature 384:372-375 (1996);Yeh et al., Immunity 7:715-725 (1997); Lee et al., Immunity 7:703-713(1997); and Kelliher et al., Immunity 8:297-303 (1998). Finally, thereexists a subsidiary death pathway involving the death domain-containingadaptor RAIDD that binds to caspase-2 and has been shown to be part ofthe TNFR1 receptor complex, although the exact physiologic relevance ofthis redundant pathway remains unclear (Duan et al., Nature 385:86-89(1997); and Ahmad et al., Cancer Res. 57:615-619 (1997).

Here, we report the identification and initial characterization of TR9,a new member of the TNF receptor family possessing a cytoplasmic deathdomain. TR9 induced apoptosis in mammalian cells and was capable ofengaging the NF κB and JNK pathways.

Materials and Methods

Expression Constructs—TR9 (amino acid residues 42-655 as depicted inFIGS. 1A-D; amino acid residues 2-615 as presented in SEQ ID NO:2) andTR9 delta (amino acid residues 42-460 as depicted in FIGS. 1A-D; aminoacid residues 2-420 as presented in SEQ ID NO:2) were cloned intopCMV1FLAG (IBI-Kodak) as in frame fusions to a TR9-terminalPreprotrypsin leader sequence and FLAG tag encoded by the vector. cDNAswere obtained by polymerase chain reaction using DNA oligo primers forTR9: 5′-GGAAGATCTGCCAGAACAGAAGGCCTCGAAT-3′ (SEQ ID NO:16) and5′-CCATCTTCCTGACCTG CTGTAGTCTAGAGCC-3′ (SEQ ID NO:17) and for TR9 delta:5′-GGAAGATCTGCCAGAACAGAAGG CCTCGAAT-3′ (SEQ ID NO:16) and 5′-GCCGACCACGAGCGGGCCTAGTCTAGAGCC-3′ (SEQ ID NO:18). Constructs encoding DR4, FADD,CD95, DR3, TRADD, ICH1-pro, RAIDD and RIP have been described previously(Chinnaiyan et al., Cell 81:505-512 (1995); Hsu et al., Cell 81:495-504(1995); Hsu et al., Immunity 4:387-396 (1996); Chinnaiyan et al.,Science 274:990-992 (1996); Kelliher et al., Immunity 8:297-303 (1998);and Pan et al., Science 276:111-113 (1997)).

Apoptosis Assay—Cell death assays were performed as previously described(Chinnaiyan et al., Cell 81:505-512 (1995); and Pan et al., Science276:111-113 (1997)). Both Hela and MCF7 cells were transfected using thelipofectamine procedure (Life Technologies, Inc.) according to themanufacturer's instructions.

Co-immunoprecipitation Assay—In vivo interaction assays have beendescribed elsewhere (Chinnaiyan et al., Cell 81:505-512 (1995); and Panet al., Science 276:111-113 (1997)). 293 cells were co-transfected withFLAG-TR9, FLAG-TR9 delta, FLAG-CD95, FLAG-DR3, FLAG-TNFR1, andICH-1pro-FLAG, expression constructs using standard calcium phosphateprecipitation. After transfection (at 38-40 hours), cell lysates wereprepared and the FLAG-tagged expressed proteins were immunoprecipitatedwith FLAG M2 affinity gel (IBI-Kodak) and the presence of FADD,myc-tagged TRADD and RIP (myc-TRADD and myc-RIP), or RAIDD detected byimmunoblotting with polyclonal antibody to FADD horseradish peroxidase(HRP)-conjugated antibody to myc (BMB), or polyclonal antibody to RAIDD.

NF-κK Luciferase Assay—NF κB luciferase assays were done as describedelsewhere (Chinnaiyan et al., Cell 81:505-512 (1995); and Pan et al.,Science 276:111-113 (1997)).

JNK Activation Assay—293 cells were cultured in MEM containing 10% FBS.Cells were plated in 6-well plates and transfected with TR9 expressingplasmid or vector alone at 60-70% confluency by the lipofectamine methodaccording to the manufacturer's instructions. Forty hours posttransfection, cell extracts were prepared in lysis buffer containing 20mM HEPES, pH 7.4, 2 mM EDTA, 250 MM NaCl, 0.1% NP-40, 2 μg/ml leupeptin,2 μg/ml aprotinin, 1 mM PMSF, 0.5 μg/ml benzamide, 1 mM DTT and 1 mMorthovanadate. The C-jun kinase assay was performed by a modified methodas described (Haridas et al., Immunol. 160:3152-3162 (1998)). Briefly,cell extracts (70 μg) were subjected to immunoprecipitation with 0.03 μganti-JNK antibody for 30 min at 4° C. Immuno-complexes were collected byincubation with protein A/G-sepharose beads for 30 min at 4° C. Thebeads were extensively washed with lysis buffer (4×400 μl) and kinasebuffer (2×400 μl: 20 mM HEPES, pH 7.4, 1 mM DTT, 25 mM NaCl) and thekinase reaction allowed to proceed for 15 min at 30° C. with 2 μgGST-Jun (1-79) in 20 μl containing 20 mM HEPES, pH 7.4, 10 mM MgCl₂ 1 mMDTT and 10 μCi [γ³²P]ATP. Reactions were stopped by the addition of 15μl SDS-sample buffer and resolved by SDS-polyacrylamide gelelectrophoresis. GST-Jun (1-79) was visualized by staining withCoomassie Blue and the dried gel visualized following Phosphorimageranalysis (Molecular Dynamics; Sunyvale, Calif.) and quantitation byImageQuant Software (Molecular Dynamics). A specific assay for JNKactivity involved the co-transfection of 3×10⁶ 293 cells with vector, orthe CD40, TR9, or TR9 delta expression constructs (6.4 μg) together with2.4 μg of a JNK-myc expression plasmid using the calcium phosphateprecipitation method. After transfection (approximately 36 hours), cellextracts were prepared by lysis in NP 40 buffer (20 mM Tris-Cl, pH 8.0,137 mM NaCl, 10% Glycerol, 2 mM EDTA, 5 mM Na₂VO₄, 0.5 mM PMSF and 1%NP40) plus protease inhibitor cocktail (BMB). Inmuunoprecipitation ofJNK-myc was performed using monoclonal anti-myc antibody (10 μg, Babco)and immunocomplexes precipitated with 20 μl protein G-sepharose (50%slurry, Sigrna) and detected by blotting with anti-myc-HRP. FLAG taggedCD40, TR9, and TR9 delta were immunoprecipitated with anti-FLAG M2affinity gel and detected by blotting with anti-FLAG antibody. Thekinase assay utilized 2 μg GST Jun(1-79) as substrate, 50 mM ATP and 5μCi γ³²P]ATP in 30 μl kinase buffer (30 mM HEPES, pH 7.4, 7 mM Mn Cl₂, 5mM MgCl₂ and 1 mM DTT).

Results and Discussion

TR9 has a putative signal sequence (amino acid residues 1-41 as depictedin FIGS. 1A-D and 4A; amino acid residues −40 to 1 in SEQ ID NO:2), withthe mature form predicted to start at amino acids 42 (Gln) as depictedin FIGS. 1A-D and 4A (Nielson et al., Protein. Eng. 10:1-6 (1997)). Theextracellular portion (amino acid residues 42-350 as depicted in FIGS.1A-D and 4A; amino acid residues 2-310 in SEQ ID NO:2) contains fourTNFR-like cysteine-rich motifs of TR9 (amino acid residues 67-211 asdepicted in FIGS. 1A-D; amino acid residues 27-171 in SEQ ID NO:2) thatare most related to those of osteoprotegerin (OPG) and TNFR2 with 36%and 42% amino acid identities, respectively (FIG. 4B; data not shown). Atransmembrane domain (amino acids 351 to 370 as depicted in FIGS. 1A-Dand 4A; residues 311 to 330 of SEQ ID NO:2) is followed by a 285-aminoacid long cytoplasmic portion of the molecule that contains a deathdomain related to those of all known death receptors (FIG. 4C), beingmost related to the death domain of TNFR1 (27.2%) and least like that ofDR5 (19.7%). Curiously, unlike other death receptors that have deathdomains present in their COOH-terminus, the death domain in TR9 waslocated adjacent to the transmembrane domain followed by a 150 aminoacid tail. Interestingly, following the death domain was a putativeleucine zipper sequence overlapping with a proline-rich regionreminiscent of a SH3 domain-binding motif (FIG. 4A) (Pawson et al.,Science 278:2075-2080 (1997)).

TR9 mRNA expression in human tissues and cancer cell lines—A 4-kb TR9transcript was found in most human adult tissue, immune tissue, andcancer cell lines represented on Northern blots (Clontech) that wereprobed with TR9 cDNA according to the manufacturers instructions (datanot shown). The transcript was abundant in heart, brain, placenta,pancreas, lymph node, thymus and prostate. Lower levels were detected inlung, skeletal muscle, kidney, testis, uterus, small intestine, colon,spleen, bone marrow, and fetal liver. However, adult liver andperipheral blood leukocytes expressed little TR9 mRNA. Additionally,smaller transcripts of 3.1 and 2.4 kb were observed in the testis andfetal liver, respectively.

Among human cancer cell lines, abundant levels of 4-kb transcript wasdetected in several nonlymphoid tumor cells, including cervicalcarcinoma Hela S3, colorectal adenocarcinoma SW480, lung carcinoma A549,and melanoma G361 cells. Significantly, less or no expression wasobserved in lines of hematopoietic origin (e.g., Raji, K562, and HL-60;data not shown).

TR9 induces apoptosis in mammalian cells—Since ectopic expression ofdeath receptors can induce cell death in a ligand-independent manner(Chinnaiyan et al., Cell 81:505-512 (1995); Boldin et al., J. Biol.Chem. 270:7795-7789 (1995); Chinnaiyan et al., Science 274:990-992(1996); Kitson et al., Nature 384:372-375 (1996); and Pan et al.,Science 276:111-113 (1997)), we tested if TR9 could induce apoptosisupon overexpression. When Hela S3 cervical carcinoma cells weretransfected with a TR9-expressing construct, 43% of the transfectedcells underwent morphological changes characteristic of apoptosis (FIG.5). As expected, deletion of the putative death domain (TR9 delta)abolished its killing activity. Significantly, TR9 was unable to inducecell death in human breast carcinoma MCF7 cells although they were verysensitive to DR4 killing (FIG. 5 and not shown), suggesting that thecell death pathway engaged by TR9 may be distinct from that engaged byother death receptors. Alternatively, the apoptotic activity of TR9 maybe modulated by other signaling pathways it activates (see below) orligand binding may be required to unveil its full killing capacity.

Interaction of TR9 with adaptor molecules in vivo—Death receptorsutilize the adaptor molecules FADD (for CD95) or both TRADD and FADD(for TNFR1 and DR3) to transmit the death signal (Chinnaiyan et al.,Cell 81:505-512 (1995); Boldin et al., J. Biol. Chem. 270:7795-7789(1995); Chinnaiyan et al., Science 274:990-992 (1996); and Kitson etal., Nature 384:372-375 (1996)). We thus determined if TR9 could bindany of these adaptor molecules in human embryonic kidney 293 cells. TR9did not interact with FADD, although the association between CD95 andFADD was readily detected under similar conditions (data not shown).Interestingly, TR9 was found to associate with TRADD, although theinteraction was weaker than that between DR3 and TRADD (data not shown).This observation is consistent with the observation that TR9 has aweaker killing ability. Alternatively, TR9 may use a TRADD-relatedmolecule as an adaptor, or the observed association might be bridged byanother adaptor protein. Interaction was not detectable between TR9 andRAIDD or RIP, two other adaptor molecules known to be recruited to theTNFR1 and DR3 signalling complexes (data not shown).

TR9 activates nuclear factor-κB—Both TNFR1 and DR3 can engage a signaltransduction pathway that leads to the activation of NF-κB (Smith etal., Cell 76: 959-962 (1994); Chinnaiyan et al., Science 274:990-992(1996); Kitson et al., Nature 384:372-375 (1996); and Baker et al.,Oncogene 12:1-9 (1996). The ability of TR9 to activate NF-κB was testedin a luciferase reporter assay and was found to induce NF-κB activationin a dose-dependent manner (FIG. 6). Presumably overexpressing thereceptor allowed it to achieve an active configuration that wascompetent to signal the NF-κB system. Interestingly, the cytoplasmicdeletion of TR9 that abolished its apoptotic activity similarlyabrogated its ability to activate NF-κB (data not shown), suggestingthat these two signaling pathways may be mediated by a commonreceptor-proximal adapter molecule.

Ectopic expression of TR9 induces JNK activation—JNK activation is knownto be induced by several TNF receptors including TNFR1 and CD40 (Smithet al., Cell 76: 959-962 (1994); Yeh et al., Immunity 7:715-725 (1997);Lee et al., Immunity 7:703-713 (1997); and Baker et al., Oncogene 12:1-9(1996)). We next determined whether overexpression of TR9 could lead toJNK activation using an in vitro kinase assay. TR9 was found to induceJNK activation in a dose-dependent manner (data not shown). Thecytoplasmic truncation that attenuated cell death or NF-κB activationhad surprisingly little effect on JNK activation (data not shown). Thiswould be consistent with the notion that JNK activation is mediated by acytoplasmic segment different from that responsible for apoptosis andNF-κB induction. It is noteworthy that two potential TRAF-binding motifsare present adjacent to the transmembrane domain PRQDP (amino acidresidues 381-385 as depicted in FIGS. 1A-D; amino acid residues 341-345as presented in SEQ ID NO:2), and PTQNR (amino acid residues 400-404 asdepicted in FIGS. 1A-D; amino acid residues 360-364 as presented in SEQID NO:2) (Gedrich et al., J. Biol. Chem. 271:12852-12858 (1996) andBoucher et al., Biochem. Biophys. Res. Commun. 233:592-600 (1997).

In conclusion, we have identified a novel death domain-containing TNFreceptor designated TR9. TR9 engages a cell death pathway different fromthose initiated by the CD95, TNFR1 or TRAIL/Apo2L receptors. Inaddition, TR9 also activates NF-κB and JNK, two signaling pathwaysshared by TNFR1. Thus, it is likely that like the other members of theTNF receptor family, TR9 plays a role in inflammatory responses andimmune regulation.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference.

1. An isolated nucleic acid molecule comprising a polynucleotide havinga nucleotide sequence at least 95% identical to a sequence selected fromthe group consisting of: (a) a nucleotide sequence encoding apolypeptide comprising amino acids from about −40 to about 615 in SEQ IDNO:2; (b) a nucleotide sequence encoding a polypeptide comprising aminoacids from about −39 to about 615 in SEQ ID NO:2; (c) a nucleotidesequence encoding a polypeptide comprising amino acids from about 1 toabout 615 in SEQ ID NO:2; (d) a nucleotide sequence encoding apolypeptide having the amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 209037; (e) a nucleotide sequence encodingthe mature TR9 polypeptide having the amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No. 209037; (f) a nucleotidesequence encoding the TR9 extracellular domain; (g) a nucleotidesequence encoding the TR9 transmembrane domain; (h) a nucleotidesequence encoding the TR9 intracellular domain; (i) a nucleotidesequence encoding the TR9 receptor extracellular and intracellulardomains with all or part of the transmembrane domain deleted; anucleotide sequence encoding the TR9 death domain; and (k) a nucleotidesequence complementary to any of the nucleotide sequences in (a), (b),(c), (d), (e), (f), (g), (h), (i), or (j).
 2. The nucleic acid moleculeof claim 1 wherein said polynucleotide has the nucleotide sequence inSEQ ID NO:1.
 3. The nucleic acid molecule of claim 1 wherein saidpolynucleotide has the nucleotide sequence in SEQ ID NO:1 encoding theTR9 receptor having the amino acid sequence in SEQ ID NO:2.
 4. Thenucleic acid molecule of claim 1 wherein said polynucleotide has thenucleotide sequence in SEQ ID NO:1 encoding the mature TR9 receptorhaving the amino acid sequence in SEQ ID NO:2.
 5. The nucleic acidmolecule of claim 1 wherein said polynucleotide has the nucleotidesequence of the cDNA clone contained in ATCC Deposit No.
 209037. 6. Thenucleic acid molecule of claim 1 wherein said polynucleotide has thenucleotide sequence encoding the TR9 receptor having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 209037.7. The nucleic acid molecule of claim 1 wherein said polynucleotide hasthe nucleotide sequence encoding the mature TR9 receptor having theamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo.
 209037. 8. An isolated nucleic acid molecule comprising apolynucleotide which hybridizes under stringent hybridization conditionsto a polynucleotide having a nucleotide sequence identical to anucleotide sequence in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j),or (k) of claim 1 wherein said polynucleotide which hybridizes does nothybridize under stringent hybridization conditions to a polynucleotidehaving a nucleotide sequence consisting of only A residues or of only Tresidues.
 9. An isolated nucleic acid molecule comprising apolynucleotide which encodes the amino acid sequence of anepitope-bearing portion of a TR9 receptor having an amino acid sequencein (a), (b), (c), (d), (e), (f), (g), (h), (i), or (j) of claim
 1. 10.The isolated nucleic acid molecule of claim 9, which encodes anepitope-bearing portion of a TR9 receptor selected from the groupconsisting of: a polypeptide comprising amino acid residues from about 4to about 81 in SEQ ID NO:2; a polypeptide comprising amino acid residuesfrom about 116 to about 271 in SEQ ID NO:2; a polypeptide comprisingamino acid residues from about 283 to about 308 in SEQ ID NO:2; apolypeptide comprising amino acid residues from about 336 to about 372in SEQ ID NO:2; a polypeptide comprising amino acid residues from about393 to about 434 in SEQ ID NO:2; a polypeptide comprising amino acidresidues from about 445 to about 559 in SEQ ID NO:2; and a polypeptidecomprising amino acid residues from about 571 to about 588 in SEQ IDNO:2.
 11. The isolated nucleic acid molecule of claim 1, which encodesthe TR9 receptor extracellular domain.
 12. The isolated nucleic acidmolecule of claim 1, which encodes the TR9 receptor transmembranedomain.
 13. The isolated nucleic acid molecule of claim 1, which encodesthe TR9 receptor intracellular domain.
 14. An isolated nucleic acidmolecule comprising a polynucleotide having a sequence at least 95%identical to a sequence selected from the group consisting of: (a) thenucleotide sequence of clone HIBEJ86R (SEQ ID NO:6); (b) the nucleotidesequence of clone HL1AA79R (SEQ ID NO:7); (c) the nucleotide sequence ofclone HHFGD57R (SEQ ID NO:8); (d) the nucleotide sequence of cloneHSABG38R (SEQ ID NO:9); (e) the nucleotide sequence of clone HHPDZ31R(SEQ ID NO:10); (f) the nucleotide sequence of a portion of the sequenceshown in SEQ ID NO:1 wherein said portion comprises at least 50contiguous nucleotides from nucleotide 500 to nucleotide 980; and (g) anucleotide sequence complementary to any of the nucleotide sequences in(a), (b), (c), (d), (e), or (f) above.
 15. A method for making arecombinant vector comprising inserting an isolated nucleic acidmolecule of claim 1 into a vector.
 16. A recombinant vector produced bythe method of claim
 15. 17. A method of making a recombinant host cellcomprising introducing the recombinant vector of claim 16 into a hostcell.
 18. A recombinant host cell produced by the method of claim 17.19. A recombinant method for producing a TR9 polypeptide, comprisingculturing the recombinant host cell of claim 18 under conditions suchthat said polypeptide is expressed and recovering said polypeptide. 20.An isolated TR9 polypeptide having an amino acid sequence at least 95%identical to a sequence selected from the group consisting of: (a) aminoacids from about −40 to about 615 in SEQ ID NO:2; (b) amino acids fromabout −39 to about 615 in SEQ ID NO:2; (c) amino acids from about 1 toabout 615 in SEQ ID NO:2; (d) the amino acid sequence of the TR9polypeptide having the amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 209037; (e) the amino acid sequence of themature TR9 polypeptide having the amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No. 209037; (f) the amino acidsequence of the TR9 receptor extracellular domain; (g) the amino acidsequence of the TR9 receptor transmembrane domain; (h) the amino acidsequence of the TR9 receptor intracellular domain; (i) the amino acidsequence of the TR9 receptor intracellular and extracellular domainswith all or part of the transmembrane domain deleted; the amino acidsequence of the TR9 receptor death domain; and (k) the amino acidsequence of an epitope-bearing portion of any one of the polypeptides of(a), (b), (c), (d), (e), (f), (g), (h), (i), or (j).
 21. An isolatedpolypeptide comprising an epitope-bearing portion of the TR9 receptorprotein, wherein said portion is selected from the group consisting of:a polypeptide comprising amino acid residues from about 4 to about 81 inSEQ ID NO:2; a polypeptide comprising amino acid residues from about 116to about 271 in SEQ ID NO:2; a polypeptide comprising amino acidresidues from about 283 to about 308 in SEQ ID NO:2; a polypeptidecomprising amino acid residues from about 336 to about 372 in SEQ IDNO:2; a polypeptide comprising amino acid residues from about 393 toabout 434 in SEQ ID NO:2; a polypeptide comprising amino acid residuesfrom about 445 to about 559 in SEQ ID NO:2; and a polypeptide comprisingamino acid residues from about 571 to about 588 in SEQ ID NO:2.
 22. Anisolated antibody that binds specifically to a TR9 receptor polypeptideof claim
 20. 23. An isolated nucleic acid molecule comprising apolynucleotide encoding a TR9 receptor polypeptide wherein, except forat least one conservative amino acid substitution, said polypeptide hasa sequence selected from the group consisting of: (a) a nucleotidesequence encoding a polypeptide comprising amino acids from about −40 toabout 615 in SEQ ID NO:2; (b) a nucleotide sequence encoding apolypeptide comprising amino acids from about −39 to about 615 in SEQ IDNO:2; (c) a nucleotide sequence encoding a polypeptide comprising aminoacids from about 1 to about 615 in SEQ ID NO:2; (d) a nucleotidesequence encoding a polypeptide having the amino acid sequence encodedby the cDNA clone contained in ATCC Deposit No. 209037; (e) a nucleotidesequence encoding the mature TR9 polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 209037;(f) a nucleotide sequence encoding the TR9 extracellular domain; (g) anucleotide sequence encoding the TR9 transmembrane domain; (h) anucleotide sequence encoding the TR9 intracellular domain; (i) anucleotide sequence encoding the TR9 receptor extracellular andintracellular domains with all or part of the transmembrane domaindeleted; (j) a nucleotide sequence encoding the TR9 death domain; and(k) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), or (j).
 24. Anisolated TR9 receptor polypeptide wherein, except for at least oneconservative amino acid substitution, said polypeptide has a sequenceselected from the group consisting of: (a) amino acids from about −40 toabout 615 in SEQ ID NO:2; (b) amino acids from about −39 to about 615 inSEQ ID NO:2; (c) amino acids from about 1 to about 615 in SEQ ID NO:2;(d) the amino acid sequence of the TR9 polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 209037;(e) the amino acid sequence of the mature TR9 polypeptide having theamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo. 209037; (f) the amino acid sequence of the TR9 receptorextracellular domain; (g) the amino acid sequence of the TR9 receptortransmembrane domain; (h) the amino acid sequence of the TR9 receptorintracellular domain; (i) the amino acid sequence of the TR9 receptorextracellular and intracellular domains with all or part of thetransmembrane domain deleted; (j) the amino acid sequence of the TR9receptor death domain; and (k) the amino acid sequence of anepitope-bearing portion of any one of the polypeptides of (a), (b), (c),(d), (e), (f), (g), (h), (i), or (j).